During 1942 and into 1943, FAA squadrons progressively converted to the Seafire, eventually replacing the Sea Hurricane in front-line service. In the Fleet Air Arm, Spitfires and Seafires were used by a number of squadrons, the Spitfires used by training and land based squadrons. Twelve 800 series squadrons used Spitfires and Seafires (Numbers 801 NAS, 802 NAS, 808 NAS, 809 NAS, 879 NAS, 880 NAS, 884 NAS, 885 NAS, 886 NAS, 887 NAS, 897 NAS and 899 NAS). Several units of the Royal Naval Volunteer Reserve also flew Seafires postwar, including 1831, 1832 and 1833 squadrons.

In November 1942, the first combat use of the Seafire occurred during in Operation Torch, the Allied landings in North Africa, from the decks of several escort and fleet carriers; unusually, Seafires flew with American star markings during the operation, these were removed following their withdrawal from the theatre.[34] In July 1943, the Seafire was used to provide air cover for the Allied invasion of Sicily; and reprised this role in September 1943 during the subsequent Allied invasion of Italy, being used to maintain continuous air cover of the beachheads, the type being almost entirely responsible for this task. During the latter operation, around half of the taskforce's Seafires were inoperable within four days, primarily due to landing accidents.

During 1944, Seafires were used for air cover in support of several aerial bombing missions against the German battleship Tirpitz. In June 1944, multiple Seafire squadrons were used during the Normandy landings for the purpose of locating in-land targets for naval gunnery batteries to attack; during this operation, these aircraft had been placed under RAF control and were operated from shore bases, these were returned to FAA control in July 1944. In August 1944, Seafires were used to support Allied ground forces during Operation Dragoon in Southern France.

During the latter half of the war, the Seafire saw increasing service as part of Britain's contribution to the Far East Pacific campaigns, serving with No. 887 and 894 Squadrons, Fleet Air Arm, aboard HMS Indefatigable and joining the British Pacific Fleet late in 1944. As range quickly became a detrimental factor in Pacific operations, Seafires in this theatre were often fitted with additional fuel tanks previously used by Curtiss P-40 Warhawks. Due to their good high altitude performance and lack of ordnance-carrying capabilities (compared to the Hellcats and Corsairs of the Fleet) the Seafires were allocated the vital defensive duties of Combat Air Patrol (CAP) over the fleet. During May 1945, Seafires were used to cover the Allied landings at Rangoon for Operation Crimson.[39] Seafires were thus heavily involved in countering the kamikaze attacks during the Okinawa landings and beyond. The Spitfire was operational in the Pacific Fleet right up to VJ Day, being used off the coast of Japan during the final months of the war.

The Seafires' best day was 15 August 1945, shooting down eight attacking aircraft for one loss. During the campaign 887 NAS claimed 12 kills and 894 NAS claimed 10 kills (with two more claims earlier in 1944 over Norway). The top scoring Seafire pilot of the war was Sub-Lieutenant R.H. Reynolds DSC of 894, who claimed 4.5 air victories in 1944-5.