diff --git a/datasette/cli.py b/datasette/cli.py
index 24d87279..94af09a2 100644
--- a/datasette/cli.py
+++ b/datasette/cli.py
@@ -590,13 +590,20 @@ def serve(
         internal=internal,
     )
 
-    # if files is a single directory, use that as config_dir=
-    if 1 == len(files) and os.path.isdir(files[0]):
-        kwargs["config_dir"] = pathlib.Path(files[0])
-        files = []
+    # Separate directories from files
+    directories = [f for f in files if os.path.isdir(f)]
+    file_paths = [f for f in files if not os.path.isdir(f)]
+
+    # Handle config_dir - only one directory allowed
+    if len(directories) > 1:
+        raise click.ClickException(
+            "Cannot pass multiple directories. Pass a single directory as config_dir."
+        )
+    elif len(directories) == 1:
+        kwargs["config_dir"] = pathlib.Path(directories[0])
 
     # Verify list of files, create if needed (and --create)
-    for file in files:
+    for file in file_paths:
         if not pathlib.Path(file).exists():
             if create:
                 sqlite3.connect(file).execute("vacuum")
@@ -607,8 +614,32 @@ def serve(
                     )
                 )
 
-    # De-duplicate files so 'datasette db.db db.db' only attaches one /db
-    files = list(dict.fromkeys(files))
+    # Check for duplicate files by resolving all paths to their absolute forms
+    # Collect all database files that will be loaded (explicit files + config_dir files)
+    all_db_files = []
+
+    # Add explicit files
+    for file in file_paths:
+        all_db_files.append((file, pathlib.Path(file).resolve()))
+
+    # Add config_dir databases if config_dir is set
+    if "config_dir" in kwargs:
+        config_dir = kwargs["config_dir"]
+        for ext in ("db", "sqlite", "sqlite3"):
+            for db_file in config_dir.glob(f"*.{ext}"):
+                all_db_files.append((str(db_file), db_file.resolve()))
+
+    # Check for duplicates
+    seen = {}
+    for original_path, resolved_path in all_db_files:
+        if resolved_path in seen:
+            raise click.ClickException(
+                f"Duplicate database file: '{original_path}' and '{seen[resolved_path]}' "
+                f"both refer to {resolved_path}"
+            )
+        seen[resolved_path] = original_path
+
+    files = file_paths
 
     try:
         ds = Datasette(files, **kwargs)
diff --git a/tests/test_cli.py b/tests/test_cli.py
index 537089ac..1c8f51ef 100644
--- a/tests/test_cli.py
+++ b/tests/test_cli.py
@@ -447,17 +447,6 @@ def test_serve_duplicate_database_names(tmpdir):
     assert {db["name"] for db in databases} == {"db", "db_2"}
 
 
-def test_serve_deduplicate_same_database_path(tmpdir):
-    "'datasette db.db db.db' should only attach one database, /db"
-    runner = CliRunner()
-    db_path = str(tmpdir / "db.db")
-    sqlite3.connect(db_path).execute("vacuum")
-    result = runner.invoke(cli, [db_path, db_path, "--get", "/-/databases.json"])
-    assert result.exit_code == 0, result.output
-    databases = json.loads(result.output)
-    assert {db["name"] for db in databases} == {"db"}
-
-
 @pytest.mark.parametrize(
     "filename", ["test-database (1).sqlite", "database (1).sqlite"]
 )
@@ -496,3 +485,57 @@ def test_internal_db(tmpdir):
     )
     assert result.exit_code == 0
     assert internal_path.exists()
+
+
+def test_duplicate_database_files_error(tmpdir):
+    """Test that passing the same database file multiple times raises an error"""
+    runner = CliRunner()
+    db_path = str(tmpdir / "test.db")
+    sqlite3.connect(db_path).execute("vacuum")
+
+    # Test with exact duplicate
+    result = runner.invoke(cli, ["serve", db_path, db_path, "--get", "/"])
+    assert result.exit_code == 1
+    assert "Duplicate database file" in result.output
+    assert "both refer to" in result.output
+
+    # Test with different paths to same file (relative vs absolute)
+    result2 = runner.invoke(
+        cli, ["serve", db_path, str(pathlib.Path(db_path).resolve()), "--get", "/"]
+    )
+    assert result2.exit_code == 1
+    assert "Duplicate database file" in result2.output
+
+    # Test that a file in the config_dir can't also be passed explicitly
+    config_dir = tmpdir / "config"
+    config_dir.mkdir()
+    config_db_path = str(config_dir / "data.db")
+    sqlite3.connect(config_db_path).execute("vacuum")
+
+    result3 = runner.invoke(
+        cli, ["serve", config_db_path, str(config_dir), "--get", "/"]
+    )
+    assert result3.exit_code == 1
+    assert "Duplicate database file" in result3.output
+    assert "both refer to" in result3.output
+
+    # Test that mixing a file NOT in the directory with a directory works fine
+    other_db_path = str(tmpdir / "other.db")
+    sqlite3.connect(other_db_path).execute("vacuum")
+
+    result4 = runner.invoke(
+        cli, ["serve", other_db_path, str(config_dir), "--get", "/-/databases.json"]
+    )
+    assert result4.exit_code == 0
+    databases = json.loads(result4.output)
+    assert {db["name"] for db in databases} == {"other", "data"}
+
+    # Test that multiple directories raise an error
+    config_dir2 = tmpdir / "config2"
+    config_dir2.mkdir()
+
+    result5 = runner.invoke(
+        cli, ["serve", str(config_dir), str(config_dir2), "--get", "/"]
+    )
+    assert result5.exit_code == 1
+    assert "Cannot pass multiple directories" in result5.output