diff --git a/CHANGELOG.md b/CHANGELOG.md
index 1f252e8290..39624e81ae 100644
--- a/CHANGELOG.md
+++ b/CHANGELOG.md
@@ -11,6 +11,9 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0
 
 ## Unreleased
 
+- `opentelemetry-instrumentation-fastapi`: Fix memory leak in `uninstrument_app()` by properly removing apps from the tracking set
+  ([#3688](https://github.com/open-telemetry/opentelemetry-python-contrib/pull/3688)
+
 ## Version 1.36.0/0.57b0 (2025-07-29)
 
 ### Fixed
@@ -1760,8 +1763,8 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0
   ([#195](https://github.com/open-telemetry/opentelemetry-python-contrib/pull/195))
 - `opentelemetry-instrumentation-dbapi` Stop capturing query parameters by default
   ([#156](https://github.com/open-telemetry/opentelemetry-python-contrib/pull/156))
-- `opentelemetry-instrumentation-asyncpg` Update asyncpg instrumentation to follow semantic conventions
-  ([#188](https://github.com/open-telemetry/opentelemetry-python-contrib/pull/188))
+- `opentelemetry-instrumentation-fastapi`: Fix memory leak in `uninstrument_app()` by properly removing apps from the tracking set
+  ([#3688](https://github.com/open-telemetry/opentelemetry-python-contrib/pull/3688))
 - `opentelemetry-instrumentation-grpc` Update protobuf versions
   ([#1356](https://github.com/open-telemetry/opentelemetry-python/pull/1356))
 
diff --git a/instrumentation/opentelemetry-instrumentation-fastapi/src/opentelemetry/instrumentation/fastapi/__init__.py b/instrumentation/opentelemetry-instrumentation-fastapi/src/opentelemetry/instrumentation/fastapi/__init__.py
index 8ba83985c6..3e39a74a49 100644
--- a/instrumentation/opentelemetry-instrumentation-fastapi/src/opentelemetry/instrumentation/fastapi/__init__.py
+++ b/instrumentation/opentelemetry-instrumentation-fastapi/src/opentelemetry/instrumentation/fastapi/__init__.py
@@ -186,6 +186,7 @@ def client_response_hook(span: Span, scope: dict[str, Any], message: dict[str, A
 import logging
 import types
 from typing import Collection, Literal
+from weakref import WeakSet as _WeakSet
 
 import fastapi
 from starlette.applications import Starlette
@@ -358,6 +359,11 @@ def uninstrument_app(app: fastapi.FastAPI):
         app.middleware_stack = app.build_middleware_stack()
         app._is_instrumented_by_opentelemetry = False
 
+        # Remove the app from the set of instrumented apps to avoid calling uninstrument twice
+        # if the instrumentation is later disabled or such
+        # Use discard to avoid KeyError if already GC'ed
+        _InstrumentedFastAPI._instrumented_fastapi_apps.discard(app)
+
     def instrumentation_dependencies(self) -> Collection[str]:
         return _instruments
 
@@ -388,7 +394,11 @@ def _instrument(self, **kwargs):
         fastapi.FastAPI = _InstrumentedFastAPI
 
     def _uninstrument(self, **kwargs):
-        for instance in _InstrumentedFastAPI._instrumented_fastapi_apps:
+        # Create a copy of the set to avoid RuntimeError during iteration
+        instances_to_uninstrument = list(
+            _InstrumentedFastAPI._instrumented_fastapi_apps
+        )
+        for instance in instances_to_uninstrument:
             self.uninstrument_app(instance)
         _InstrumentedFastAPI._instrumented_fastapi_apps.clear()
         fastapi.FastAPI = self._original_fastapi
@@ -406,7 +416,8 @@ class _InstrumentedFastAPI(fastapi.FastAPI):
     _http_capture_headers_sanitize_fields: list[str] | None = None
     _exclude_spans: list[Literal["receive", "send"]] | None = None
 
-    _instrumented_fastapi_apps = set()
+    # Track instrumented app instances using weak references to avoid GC leaks
+    _instrumented_fastapi_apps = _WeakSet()
     _sem_conv_opt_in_mode = _StabilityMode.DEFAULT
 
     def __init__(self, *args, **kwargs):
@@ -426,10 +437,6 @@ def __init__(self, *args, **kwargs):
         )
         _InstrumentedFastAPI._instrumented_fastapi_apps.add(self)
 
-    def __del__(self):
-        if self in _InstrumentedFastAPI._instrumented_fastapi_apps:
-            _InstrumentedFastAPI._instrumented_fastapi_apps.remove(self)
-
 
 def _get_route_details(scope):
     """
diff --git a/instrumentation/opentelemetry-instrumentation-fastapi/tests/test_fastapi_instrumentation.py b/instrumentation/opentelemetry-instrumentation-fastapi/tests/test_fastapi_instrumentation.py
index 523c165f85..0a4fd6c037 100644
--- a/instrumentation/opentelemetry-instrumentation-fastapi/tests/test_fastapi_instrumentation.py
+++ b/instrumentation/opentelemetry-instrumentation-fastapi/tests/test_fastapi_instrumentation.py
@@ -14,7 +14,9 @@
 
 # pylint: disable=too-many-lines
 
+import gc as _gc
 import unittest
+import weakref as _weakref
 from contextlib import ExitStack
 from timeit import default_timer
 from unittest.mock import Mock, call, patch
@@ -1400,6 +1402,16 @@ def test_mark_span_internal_in_presence_of_span_from_other_framework(self):
         )
 
 
+class TestFastAPIGarbageCollection(unittest.TestCase):
+    def test_fastapi_app_is_collected_after_instrument(self):
+        app = fastapi.FastAPI()
+        otel_fastapi.FastAPIInstrumentor().instrument_app(app)
+        app_ref = _weakref.ref(app)
+        del app
+        _gc.collect()
+        self.assertIsNone(app_ref())
+
+
 @patch.dict(
     "os.environ",
     {