MacDill AFB, originally Southeast Air Base, was established, constructed, and dedicated from 1939 to early 1941, starting operational service as MacDill Field more than six months before the US entered World War Two. Before the end of construction the base was named for Colonel Leslie MacDill, a World War One pilot and aerial gunnery school commander, killed in 1938 due to engine failure.

MacDill Field was one of several Army Airfields in the Tampa Bay area in World War Two, all of which were organized under the Third Air Force, with commands split among the various fields. The main mission of MacDill was to establish US air security over the Gulf of Mexico, especially defense of US oil tankers from ports of Texas and Louisiana; the US was providing Lend-Lease equipment to the United Kingdom and Soviet Union at this time, and much of the material was being shipped from Gulf ports; there was concern that German U-boats could slip into or even through the Florida Straits and run rampant in the event of war. Furthermore, the US controlled the Panama Canal, and Florida was the last major staging base on the way to Panama. MacDill was constructed in haste, with flight operations beginning while personnel camped in tents in the buggy Florida wetlands.

MacDill units included pursuit fighters for scramble response, and reconnaissance bomber squadrons, mainly B-26 Marauders, which had good loiter time and could carry sizeable ordnance loads for on-the-spot attacks, but suffered from landing difficulties. Early in the war, MacDill was a logistics hub for Project X, a supply program shipping combat aircraft to the Philippines via the South Atlantic, across Africa and through India (which was less dangerous than over the Pacific and through Japanese controlled airspace).

As the war progressed, MacDill became an excellent location for wartime movies, standing in for Pacific tropical locations, and two Hollywood films were partly shot here, A Guy Name Joe, and Air Force. Air Force featured repainted B-26s standing in for Japanese bombers; these scenes were shot near MacDill. Memos were circulated, and Japan was certainly nowhere near Florida, but vigilant and well intentioned Coast Guard units fired on the Japanese marked planes, with no casualties. Ironically, these exterior filming shots were done at Tampa Bay because of fear that Japanese marked planes would cause panic on the West Coast.

Later in the war, MacDill converted to a mainly training mission, focusing on the B-17 Flying Fortress, and later the B-29 Superfortress. The end of the war led to rapid demobilization, and many bases inactivated, but MacDill, with a regionally important location, remained open at lower strength.

In 1946 MacDill was assigned to Strategic Air Command, and units from MacDill were sent to strategic bombing campaigns in the Korean War. The Cold War demanded a high level of alertness, and although Tampa Bay was nowhere near approaches to the Soviet Union, it was still a perfectly useful training facility and well back from the expected northern air theaters. In 1955 MacDill was again used for backdrops and exterior shots for a Hollywood film, Strategic Air Command, starring James Stewart and June Allyson. Stewart plays an Air Force Reservist called to active duty, who struggles with his wife's (Allyson) displeasure over his situation; Steward was an actual Air Force Reservist and pilot, at the time a Colonel.

By 1960 budget concerns and the ever-changing global situation made MacDill look like a surplus base; the Cuban Missile Crisis shortly put an end to this idea, and MacDill's squadrons were flown in support of the Cuban Blockade. MacDill went from apparently surplus to central for Gulf-Caribbean security, and a new unit command concept, the US Strike Command was established at MacDill for global crisis response. Over the course of the 1960s MacDill transitioned to a fighter base, transferring to Tactical Air Command in 1962, a mission the base would follow to the end of the Cold War.

When the Cold War did end, MacDill's mission was reevaluated, and a review concluded that air traffic in the Tampa Bay area was becoming too congested for safety; MacDill stayed open but its mission altered to airlift and flight operations reduced. The airlift mission has since supported the 1994-1995 Operation Support Democracy (overthrowing a revolutionary dictatorship in Haiti). The early 2000s saw another round of base realignment affecting MacDill, notably the reassignment of additional air refuelling wings and reassignment of the US Marine Forces Central Command headquarters to MacDill AFB.