diff --git a/samples/bluetooth/hci_uart_3wire/README.rst b/samples/bluetooth/hci_uart_3wire/README.rst
index df99c17c0a846..ccf412b031d60 100644
--- a/samples/bluetooth/hci_uart_3wire/README.rst
+++ b/samples/bluetooth/hci_uart_3wire/README.rst
@@ -146,11 +146,6 @@ You can use following targets:
 Check the :zephyr:code-sample:`ble_direction_finding_connectionless_rx` and the
 :zephyr:code-sample:`ble_direction_finding_connectionless_tx` for more details.
 
-Using a USB CDC ACM UART
-========================
-
-The sample can be configured to use a USB UART instead. See :zephyr_file:`samples/bluetooth/hci_uart_3wire/boards/nrf52840dongle_nrf52840.conf`.
-
 Using the controller with the Zephyr host
 =========================================
 
diff --git a/samples/bluetooth/hci_uart_3wire/boards/nrf52840dongle_nrf52840.conf b/samples/bluetooth/hci_uart_3wire/boards/nrf52840dongle_nrf52840.conf
deleted file mode 100644
index f87530dd3c2c7..0000000000000
--- a/samples/bluetooth/hci_uart_3wire/boards/nrf52840dongle_nrf52840.conf
+++ /dev/null
@@ -1,4 +0,0 @@
-CONFIG_USB_DEVICE_STACK=y
-CONFIG_USB_DEVICE_PRODUCT="Zephyr HCI UART 3Wire sample"
-CONFIG_USB_CDC_ACM=y
-CONFIG_USB_DEVICE_INITIALIZE_AT_BOOT=n
diff --git a/samples/bluetooth/hci_uart_3wire/src/main.c b/samples/bluetooth/hci_uart_3wire/src/main.c
index 5454b0cd9c7c6..7cfbe39dc3e07 100644
--- a/samples/bluetooth/hci_uart_3wire/src/main.c
+++ b/samples/bluetooth/hci_uart_3wire/src/main.c
@@ -20,8 +20,6 @@
 #include <zephyr/init.h>
 #include <zephyr/drivers/uart.h>
 
-#include <zephyr/usb/usb_device.h>
-
 #include <zephyr/net_buf.h>
 #include <zephyr/bluetooth/bluetooth.h>
 #include <zephyr/bluetooth/l2cap.h>
@@ -751,13 +749,6 @@ static int hci_uart_init(void)
 {
 	LOG_DBG("");
 
-	if (IS_ENABLED(CONFIG_USB_CDC_ACM)) {
-		if (usb_enable(NULL)) {
-			LOG_ERR("Failed to enable USB");
-			return -EINVAL;
-		}
-	}
-
 	if (!device_is_ready(h5_dev)) {
 		LOG_ERR("HCI UART %s is not ready", h5_dev->name);
 		return -EINVAL;