diff --git a/sanic/compat.py b/sanic/compat.py
index 880ca68199..4ea2ed91ff 100644
--- a/sanic/compat.py
+++ b/sanic/compat.py
@@ -3,6 +3,8 @@
 import signal
 import sys
 
+from typing import Awaitable
+
 from multidict import CIMultiDict  # type: ignore
 
 
@@ -51,7 +53,7 @@ def get_all(self, key: str):
 if use_trio:  # pragma: no cover
     import trio  # type: ignore
 
-    def stat_async(path):
+    def stat_async(path) -> Awaitable[os.stat_result]:
         return trio.Path(path).stat()
 
     open_async = trio.open_file
diff --git a/sanic/response.py b/sanic/response.py
index 4b647003a7..b1765ed141 100644
--- a/sanic/response.py
+++ b/sanic/response.py
@@ -5,7 +5,7 @@
 from functools import partial
 from mimetypes import guess_type
 from os import path
-from pathlib import Path, PurePath
+from pathlib import PurePath
 from time import time
 from typing import (
     TYPE_CHECKING,
@@ -22,7 +22,7 @@
 )
 from urllib.parse import quote_plus
 
-from sanic.compat import Header, open_async
+from sanic.compat import Header, open_async, stat_async
 from sanic.constants import DEFAULT_HTTP_CONTENT_TYPE
 from sanic.cookies import CookieJar
 from sanic.exceptions import SanicException, ServerError
@@ -340,9 +340,10 @@ async def file(
         )
 
     if isinstance(last_modified, datetime):
-        last_modified = last_modified.timestamp()
+        last_modified = last_modified.replace(microsecond=0).timestamp()
     elif isinstance(last_modified, Default):
-        last_modified = Path(location).stat().st_mtime
+        stat = await stat_async(location)
+        last_modified = stat.st_mtime
 
     if last_modified:
         headers.setdefault(