In 1780, Preble was appointed as an acting midshipman on board the Massachusetts State Navy frigate Protector "thanks in large part to his father's influence".

The frigate spent most of the year cruising off New England and the West Indies; on May 26, 1780, Protector destroyed the British armed merchantman Admiral Duff and captured 55 survivors, who infected Preble and his crewmates with fever.

During his time onboard Winthrop, Preble led a boarding party which captured a British brig near Penobscot Bay.

[4] Fifteen years of merchant service followed his Revolutionary War career and, in April 1798, he was commissioned as a lieutenant in the United States Navy.

In January 1799, he assumed command of the 14-gun brig USS Pickering and took her to the West Indies to protect American commerce during the Quasi-War with France.

Not wanting to lose such an experienced and capable officer, the secretary decided to put Preble on indefinite sick leave until a good post could be found for him.

[6] In a letter dated 13 April, 1802 Preble asked for a furlough due to a rapid decline in his health since arriving in New York in January.

[8] During this time, the United States was engaged in naval warfare with the city-state of Tripoli, whose corsairs were causing havoc amongst American merchantmen in the Mediterranean.

When he finally did arrive at Tripoli, he tried to play diplomat and sue for peace, which destabilized the strong negotiating position the Americans had been building up to that point.

Bypassing several senior officers, Preble, who was in Boston supervising the construction of a new Brig for the navy was ordered in a letter dated 14 May, 1803 to take command also of, and ready, USS Constitution for duty in the Mediterranean.

Stephen Decatur, William Bainbridge, Charles Stewart, Isaac Hull, Thomas Macdonough, James Lawrence, and David Porter served under his command at Tripoli.

[4] While commanding in Tripoli, Preble masterminded the burning of USS Philadelphia by Lieutenant Stephen Decatur on February 16, 1804, preventing the captured frigate from falling into enemy hands.

Future sea captains such as Decatur, Lawrence, and Porter took his procedures to heart at a time when the US Navy was highly unregulated.

The officers serving under him during his career also went on to become influential in the Navy Department after his death, and together they proudly wore the unofficial title of "Preble's Boys".

He returned to the United States in February 1805 and became engaged in the comparably light duty of shipbuilding activities at Portland, Maine.

By congressional resolution in March 1805, a gold medal was struck and presented to Commodore Preble for the "gallantry and good conduct" of himself and his squadron at Tripoli.