diff --git a/sanic/response/convenience.py b/sanic/response/convenience.py
index 429b3214a4..d8a3059773 100644
--- a/sanic/response/convenience.py
+++ b/sanic/response/convenience.py
@@ -148,7 +148,26 @@ async def validate_file(
         last_modified = datetime.fromtimestamp(
             float(last_modified), tz=timezone.utc
         ).replace(microsecond=0)
-    if last_modified <= if_modified_since:
+
+    if (
+        last_modified.utcoffset() is None
+        and if_modified_since.utcoffset() is not None
+    ):
+        logger.warning(
+            "Cannot compare tz-aware and tz-naive datetimes. To avoid "
+            "this conflict Sanic is converting last_modified to UTC."
+        )
+        last_modified.replace(tzinfo=timezone.utc)
+    elif (
+        last_modified.utcoffset() is not None
+        and if_modified_since.utcoffset() is None
+    ):
+        logger.warning(
+            "Cannot compare tz-aware and tz-naive datetimes. To avoid "
+            "this conflict Sanic is converting if_modified_since to UTC."
+        )
+        if_modified_since.replace(tzinfo=timezone.utc)
+    if last_modified.timestamp() <= if_modified_since.timestamp():
         return HTTPResponse(status=304)
 
 
diff --git a/tests/test_response_file.py b/tests/test_response_file.py
new file mode 100644
index 0000000000..366e613e8d
--- /dev/null
+++ b/tests/test_response_file.py
@@ -0,0 +1,55 @@
+from datetime import datetime, timezone
+from logging import INFO
+
+import pytest
+
+from sanic.compat import Header
+from sanic.response.convenience import validate_file
+
+
+@pytest.mark.parametrize(
+    "ifmod,lastmod,expected",
+    (
+        ("Sat, 01 Apr 2023 00:00:00 GMT", 1672524000, None),
+        (
+            "Sat, 01 Apr 2023 00:00:00",
+            1672524000,
+            "converting if_modified_since",
+        ),
+        (
+            "Sat, 01 Apr 2023 00:00:00 GMT",
+            datetime(2023, 1, 1, 0, 0, 0),
+            "converting last_modified",
+        ),
+        (
+            "Sat, 01 Apr 2023 00:00:00",
+            datetime(2023, 1, 1, 0, 0, 0),
+            None,
+        ),
+        (
+            "Sat, 01 Apr 2023 00:00:00 GMT",
+            datetime(2023, 1, 1, 0, 0, 0).replace(tzinfo=timezone.utc),
+            None,
+        ),
+        (
+            "Sat, 01 Apr 2023 00:00:00",
+            datetime(2023, 1, 1, 0, 0, 0).replace(tzinfo=timezone.utc),
+            "converting if_modified_since",
+        ),
+    ),
+)
+@pytest.mark.asyncio
+async def test_file_timestamp_validation(
+    lastmod, ifmod, expected, caplog: pytest.LogCaptureFixture
+):
+    headers = Header([["If-Modified-Since", ifmod]])
+
+    with caplog.at_level(INFO):
+        response = await validate_file(headers, lastmod)
+    assert response.status == 304
+    records = caplog.records
+    if not expected:
+        assert len(records) == 0
+    else:
+        record = records[0]
+        assert expected in record.message