diff --git a/src/wp-includes/block-supports/custom-css.php b/src/wp-includes/block-supports/custom-css.php
index 9d5b13426f4ef..b825695c41de3 100644
--- a/src/wp-includes/block-supports/custom-css.php
+++ b/src/wp-includes/block-supports/custom-css.php
@@ -50,6 +50,18 @@ function wp_render_custom_css_support_styles( $parsed_block ) {
 		 * and can override global styles.
 		 */
 		wp_register_style( 'wp-block-custom-css', false, array( 'global-styles' ) );
+
+		// Skip if already enqueued — prevents duplication inside Query Loop.
+		global $wp_styles;
+		$existing_inline_styles = $wp_styles->get_data( 'wp-block-custom-css', 'after' );
+		if ( is_array( $existing_inline_styles ) ) {
+			foreach ( $existing_inline_styles as $style ) {
+				if ( str_contains( $style, $selector ) ) {
+					return $parsed_block;
+				}
+			}
+		}
+
 		wp_add_inline_style( 'wp-block-custom-css', $processed_css );
 	}
 
diff --git a/tests/phpunit/tests/block-supports/wpRenderCustomCssSupportStyles.php b/tests/phpunit/tests/block-supports/wpRenderCustomCssSupportStyles.php
index 0048b379f8f33..bdd47df0520a5 100644
--- a/tests/phpunit/tests/block-supports/wpRenderCustomCssSupportStyles.php
+++ b/tests/phpunit/tests/block-supports/wpRenderCustomCssSupportStyles.php
@@ -264,4 +264,51 @@ public function data_does_not_add_class_name() {
 			),
 		);
 	}
+
+	/**
+	 * Tests that CSS is not duplicated when the same block renders multiple times (e.g. inside a Query Loop).
+	 *
+	 * @ticket 65268
+	 *
+	 * @covers ::wp_render_custom_css_support_styles
+	 */
+	public function test_css_not_duplicated_inside_query_loop() {
+		$this->test_block_name = 'test/custom-css-query-loop';
+		register_block_type(
+			$this->test_block_name,
+			array(
+				'api_version' => 3,
+				'attributes'  => array(
+					'style' => array(
+						'type' => 'object',
+					),
+				),
+				'supports'    => array( 'customCSS' => true ),
+			)
+		);
+
+		$parsed_block = array(
+			'blockName' => 'test/custom-css-query-loop',
+			'attrs'     => array(
+				'style' => array(
+					'css' => 'color: fuchsia;',
+				),
+			),
+		);
+
+		// Simulate the same block rendering 3 times inside a Query Loop.
+		wp_render_custom_css_support_styles( $parsed_block );
+		wp_render_custom_css_support_styles( $parsed_block );
+		wp_render_custom_css_support_styles( $parsed_block );
+
+		// Get the inline styles registered for wp-block-custom-css.
+		global $wp_styles;
+		$inline_css = $wp_styles->get_data( 'wp-block-custom-css', 'after' );
+
+		// CSS should appear only once, not 3 times.
+		$this->assertIsArray( $inline_css, 'Inline styles should be registered.' );
+		$combined = implode( '', $inline_css );
+		$count    = substr_count( $combined, 'color: fuchsia' );
+		$this->assertSame( 1, $count, 'CSS should only be enqueued once even when block renders multiple times.' );
+	}
 }