diff --git a/ava.config.mjs b/ava.config.mjs
index ba42f7c78..c4b841c21 100644
--- a/ava.config.mjs
+++ b/ava.config.mjs
@@ -11,6 +11,7 @@ const rewritePaths = Object.fromEntries(
 export default {
   files: ["packages/*/test/**/*.spec.ts"],
   nodeArguments: ["--no-warnings", "--experimental-vm-modules"],
+  require: ["./packages/miniflare/test/setup.mjs"],
   workerThreads: inspector.url() === undefined,
   typescript: {
     compile: false,
diff --git a/packages/miniflare/src/index.ts b/packages/miniflare/src/index.ts
index b764a8d60..0b366c045 100644
--- a/packages/miniflare/src/index.ts
+++ b/packages/miniflare/src/index.ts
@@ -449,6 +449,15 @@ function safeReadableStreamFrom(iterable: AsyncIterable<Uint8Array>) {
   );
 }
 
+// Maps `Miniflare` instances to stack traces for thier construction. Used to identify un-`dispose()`d instances.
+let maybeInstanceRegistry:
+  | Map<Miniflare, string /* constructionStack */>
+  | undefined;
+/** @internal */
+export function _initialiseInstanceRegistry() {
+  return (maybeInstanceRegistry = new Map());
+}
+
 export class Miniflare {
   readonly #gatewayFactories: PluginGatewayFactories;
   readonly #routers: PluginRouters;
@@ -498,6 +507,15 @@ export class Miniflare {
     const [sharedOpts, workerOpts] = validateOptions(opts);
     this.#sharedOpts = sharedOpts;
     this.#workerOpts = workerOpts;
+
+    // Add to registry after initial options validation, before any servers/
+    // child processes are started
+    if (maybeInstanceRegistry !== undefined) {
+      const object = { name: "Miniflare", stack: "" };
+      Error.captureStackTrace(object, Miniflare);
+      maybeInstanceRegistry.set(this, object.stack);
+    }
+
     this.#log = this.#sharedOpts.core.log ?? new NoOpLog();
     this.#timers = this.#sharedOpts.core.timers ?? defaultTimers;
     this.#host = this.#sharedOpts.core.host ?? "127.0.0.1";
@@ -1256,6 +1274,10 @@ export class Miniflare {
       await this.#stopLoopbackServer();
       // `rm -rf ${#tmpPath}`, this won't throw if `#tmpPath` doesn't exist
       await fs.promises.rm(this.#tmpPath, { force: true, recursive: true });
+
+      // Remove from instance registry as last step in `finally`, to make sure
+      // all dispose steps complete
+      maybeInstanceRegistry?.delete(this);
     }
   }
 }
diff --git a/packages/miniflare/test/index.spec.ts b/packages/miniflare/test/index.spec.ts
index 7c04de59e..a86288369 100644
--- a/packages/miniflare/test/index.spec.ts
+++ b/packages/miniflare/test/index.spec.ts
@@ -528,7 +528,7 @@ test("Miniflare: accepts https requests", async (t) => {
   t.assert(log.logs[0][1].startsWith("Ready on https://"));
 });
 
-test("Miniflare: Manually triggered scheduled events", async (t) => {
+test("Miniflare: manually triggered scheduled events", async (t) => {
   const log = new TestLog(t);
 
   const mf = new Miniflare({
@@ -545,6 +545,7 @@ test("Miniflare: Manually triggered scheduled events", async (t) => {
       }
     }`,
   });
+  t.teardown(() => mf.dispose());
 
   let res = await mf.dispatchFetch("http://localhost");
   t.is(await res.text(), "false");
diff --git a/packages/miniflare/test/plugins/core/errors/index.spec.ts b/packages/miniflare/test/plugins/core/errors/index.spec.ts
index fccc3ac29..db8271c1d 100644
--- a/packages/miniflare/test/plugins/core/errors/index.spec.ts
+++ b/packages/miniflare/test/plugins/core/errors/index.spec.ts
@@ -133,6 +133,7 @@ addEventListener("fetch", (event) => {
       },
     ],
   });
+  t.teardown(() => mf.dispose());
 
   // Check service-workers source mapped
   let error = await t.throwsAsync(mf.dispatchFetch("http://localhost"), {
diff --git a/packages/miniflare/test/plugins/core/proxy/client.spec.ts b/packages/miniflare/test/plugins/core/proxy/client.spec.ts
index 277ab0117..e610450ae 100644
--- a/packages/miniflare/test/plugins/core/proxy/client.spec.ts
+++ b/packages/miniflare/test/plugins/core/proxy/client.spec.ts
@@ -35,6 +35,8 @@ test("ProxyClient: supports service bindings with WebSockets", async (t) => {
       },
     },
   });
+  t.teardown(() => mf.dispose());
+
   const { CUSTOM } = await mf.getBindings<{
     CUSTOM: ReplaceWorkersTypes<Fetcher>;
   }>();
@@ -53,6 +55,8 @@ test("ProxyClient: supports service bindings with WebSockets", async (t) => {
 
 test("ProxyClient: supports serialising multiple ReadableStreams, Blobs and Files", async (t) => {
   const mf = new Miniflare({ script: nullScript });
+  t.teardown(() => mf.dispose());
+
   const client = await mf._getProxyClient();
   const IDENTITY = client.env.IDENTITY as {
     asyncIdentity<Args extends any[]>(...args: Args): Promise<Args>;
@@ -130,6 +134,8 @@ test("ProxyClient: poisons dependent proxies after setOptions()/dispose()", asyn
 });
 test("ProxyClient: logging proxies provides useful information", async (t) => {
   const mf = new Miniflare({ script: nullScript });
+  t.teardown(() => mf.dispose());
+
   const caches = await mf.getCaches();
   const inspectOpts: util.InspectOptions = { colors: false };
   t.is(
@@ -160,6 +166,7 @@ test("ProxyClient: stack traces don't include internal implementation", async (t
     // https://developers.cloudflare.com/workers/configuration/compatibility-dates/#do-not-throw-from-async-functions
     compatibilityFlags: ["capture_async_api_throws"],
   });
+  t.teardown(() => mf.dispose());
 
   const ns = await mf.getDurableObjectNamespace("OBJECT");
   const caches = await mf.getCaches();
@@ -189,6 +196,8 @@ test("ProxyClient: stack traces don't include internal implementation", async (t
 });
 test("ProxyClient: can access ReadableStream property multiple times", async (t) => {
   const mf = new Miniflare({ script: nullScript, r2Buckets: ["BUCKET"] });
+  t.teardown(() => mf.dispose());
+
   const bucket = await mf.getR2Bucket("BUCKET");
   await bucket.put("key", "value");
   const objectBody = await bucket.get("key");
@@ -198,6 +207,8 @@ test("ProxyClient: can access ReadableStream property multiple times", async (t)
 });
 test("ProxyClient: returns empty ReadableStream synchronously", async (t) => {
   const mf = new Miniflare({ script: nullScript, r2Buckets: ["BUCKET"] });
+  t.teardown(() => mf.dispose());
+
   const bucket = await mf.getR2Bucket("BUCKET");
   await bucket.put("key", "");
   const objectBody = await bucket.get("key");
diff --git a/packages/miniflare/test/plugins/queues/index.spec.ts b/packages/miniflare/test/plugins/queues/index.spec.ts
index 23d441139..970cdfe5b 100644
--- a/packages/miniflare/test/plugins/queues/index.spec.ts
+++ b/packages/miniflare/test/plugins/queues/index.spec.ts
@@ -66,6 +66,8 @@ test("flushes partial and full batches", async (t) => {
       },
     ],
   });
+  t.teardown(() => mf.dispose());
+
   async function send(message: unknown) {
     await mf.dispatchFetch("http://localhost/send", {
       method: "POST",
@@ -255,6 +257,7 @@ test("sends all structured cloneable types", async (t) => {
       },
     ],
   });
+  t.teardown(() => mf.dispose());
 
   await mf.dispatchFetch("http://localhost");
   timers.timestamp += 1000;
@@ -326,6 +329,8 @@ test("retries messages", async (t) => {
       }
     }`,
   });
+  t.teardown(() => mf.dispose());
+
   async function sendBatch(...messages: string[]) {
     await mf.dispatchFetch("http://localhost", {
       method: "POST",
@@ -546,6 +551,8 @@ test("moves to dead letter queue", async (t) => {
       }
     }`,
   });
+  t.teardown(() => mf.dispose());
+
   async function sendBatch(...messages: string[]) {
     await mf.dispatchFetch("http://localhost", {
       method: "POST",
@@ -648,6 +655,8 @@ test("operations permit strange queue names", async (t) => {
       }
     }`,
   });
+  t.teardown(() => mf.dispose());
+
   await mf.dispatchFetch("http://localhost");
   timers.timestamp += 1000;
   await timers.waitForTasks();
@@ -718,6 +727,8 @@ test("supports message contentTypes", async (t) => {
   },
 };`,
   });
+  t.teardown(() => mf.dispose());
+
   const res = await mf.dispatchFetch("http://localhost");
   await res.arrayBuffer(); // (drain)
   timers.timestamp += 1000;
diff --git a/packages/miniflare/test/setup.mjs b/packages/miniflare/test/setup.mjs
new file mode 100644
index 000000000..72677b9c2
--- /dev/null
+++ b/packages/miniflare/test/setup.mjs
@@ -0,0 +1,23 @@
+import { _initialiseInstanceRegistry } from "miniflare";
+
+const registry = _initialiseInstanceRegistry();
+const bigSeparator = "=".repeat(80);
+const separator = "-".repeat(80);
+
+// `process.on("exit")` is more like `worker_thread.on(`exit`)` here. It will
+// be called once AVA's finished running tests and `after` hooks. Note we can't
+// use an `after` hook here, as that would run before `miniflareTest`'s
+// `after` hooks to dispose their `Miniflare` instances.
+process.on("exit", () => {
+  if (registry.size === 0) return;
+
+  // If there are Miniflare instances that weren't disposed, throw
+  const s = registry.size === 1 ? "" : "s";
+  const was = registry.size === 1 ? "was" : "were";
+  const message = `Found ${registry.size} Miniflare instance${s} that ${was} not dispose()d`;
+  const stacks = Array.from(registry.values()).join(`\n${separator}\n`);
+  console.log(
+    [bigSeparator, message, separator, stacks, bigSeparator].join("\n")
+  );
+  throw new Error(message);
+});
diff --git a/packages/miniflare/test/test-shared/miniflare.ts b/packages/miniflare/test/test-shared/miniflare.ts
index d15572826..c3449d3b2 100644
--- a/packages/miniflare/test/test-shared/miniflare.ts
+++ b/packages/miniflare/test/test-shared/miniflare.ts
@@ -149,6 +149,6 @@ export function miniflareTest<
       t.context.mf.setOptions({ ...userOpts, ...opts } as MiniflareOptions);
     t.context.url = await t.context.mf.ready;
   });
-  test.after((t) => t.context.mf.dispose());
+  test.after.always((t) => t.context.mf.dispose());
   return test;
 }