Dorado's sea trials proved the readiness of the crew, and she sailed from Naval Submarine Base New London in Groton, Connecticut, on 6 October 1943 for the Panama Canal Zone.

However, the crew of a PBM Mariner flying boat of Patrol Squadron 210 (VP-210) based at Guantanamo Bay Naval Base in Guantánamo Bay, Cuba, assigned to provide air coverage on the evening of 12 October 1943 had received an incorrect description of the restriction area, 11 nmi (20 km; 13 mi) out of place.

At 2049 local time, under a moonlit but stormy sky, that plane attacked an unidentified submarine that it believed was outside the restriction area with three Mark-47 depth charges and a 100 lb (45 kg) Mark-4 Mod-4 demolition bomb.

Subsequently, the Board of Investigation held in Guantánamo Bay, and the more formal Naval Board of Inquiry held at the Washington Navy Yard in Washington, D.C., found that the "widely scattered oil slicks" were actually oleous in nature and not bunker oil or diesel fuel - most probably rotting vegetation like seaweed.

The second submarine, attacked by the Mariner two hours later, was certainly U-214; her log book, captured after World War II, describes firing at the aircraft.

Before she was lost, the American painter Thomas Hart Benton sailed aboard Dorado on her shakedown cruise, using that experience as the basis for his paintings Score Another for the Subs, In Slumber Deep, and The Claustrophobic Confines.