diff --git a/pkg/filelocker/filelocker.go b/pkg/filelocker/filelocker.go
index 3692bade1..13e9005e8 100644
--- a/pkg/filelocker/filelocker.go
+++ b/pkg/filelocker/filelocker.go
@@ -18,6 +18,8 @@ package filelocker
 
 import (
 	"context"
+	"errors"
+	"io/fs"
 	"os"
 	"path/filepath"
 	"time"
@@ -104,6 +106,17 @@ func (lock fileUploadLock) Lock(ctx context.Context, requestRelease func()) erro
 				continue
 			}
 		}
+		// If the upload ID uses a folder structure (e.g. projectA/upload1), the directory
+		// (e.g. projectA) might not exist, if no such upload exists already. In those cases,
+		// we just return ErrNotFound because no such upload exists anyways.
+		// TODO: This assumes that filelocker is used mostly with filestore, which is likely
+		// true, but does not have to be. If another storage backend is used, we cannot make
+		// any assumption about the folder structure. As an alternative, we should consider
+		// normalizing the upload ID to remove the folder structure as well an turn projectA/upload1
+		// into projectA-upload1.
+		if errors.Is(err, fs.ErrNotExist) {
+			return handler.ErrNotFound
+		}
 		if err != lockfile.ErrBusy {
 			// If we get something different than ErrBusy, bubble the error up.
 			return err
diff --git a/pkg/filelocker/filelocker_test.go b/pkg/filelocker/filelocker_test.go
index bd288a58f..f561d65a5 100644
--- a/pkg/filelocker/filelocker_test.go
+++ b/pkg/filelocker/filelocker_test.go
@@ -60,7 +60,7 @@ func TestFileLocker_Timeout(t *testing.T) {
 	assertEmptyDirectory(dir, a)
 }
 
-func TestMemoryLocker_RequestUnlock(t *testing.T) {
+func TestFileLocker_RequestUnlock(t *testing.T) {
 	a := assert.New(t)
 
 	dir, err := os.MkdirTemp("", "tusd-file-locker")
@@ -95,6 +95,26 @@ func TestMemoryLocker_RequestUnlock(t *testing.T) {
 	assertEmptyDirectory(dir, a)
 }
 
+func TestFileLocker_DirectoryNotFound(t *testing.T) {
+	a := assert.New(t)
+
+	dir, err := os.MkdirTemp("", "tusd-file-locker")
+	a.NoError(err)
+
+	locker := New(dir)
+
+	// The upload ID uses a directory structure, but the corresponding directories do
+	// not exist. Since there can also exist no info file in this folder, we expect
+	// the locker to return ErrNotFound
+	lock, err := locker.NewLock("nested/folder/structure/upload")
+	a.Nil(err)
+
+	err = lock.Lock(context.Background(), func() {
+		panic("must not be called")
+	})
+	a.Equal(handler.ErrNotFound, err)
+}
+
 func assertEmptyDirectory(dir string, a *assert.Assertions) {
 	file, err := os.Open(dir)
 	a.NoError(err)