What is today NAS Pensacola was originally the Pensacola Navy Yard, established 1826 as the newest Navy base in the new US Florida Territory. The new base was for new ship construction and the home port for the West Indies Squadron, which actively suppressed piracy in the Gulf of Mexico and Caribbean, as well as the international slave trade (illegal after 1807) as well as smuggling and general customs duties. At the start of the Civil War, Pensacola Navy Yard was seized by Confederate forces. When the Union won New Orleans, and were expected to sweep the region, the Confederates burned the Yard down rather than let it fall into Union hands.

In 1914 Pensacola became the first dedicated Naval Aviation Station (the other airfield, NAS North Island, was a shared Army-Navy facility). The Navy had only recently been introduced to aviation, and the concept of the carrier had only been proven in 1911. Pensacola remained the only dedicated air station, with only 38 aviators, as the US entered World War One, and the primary aviation training station.By the end of the First World War, the Navy had over 1,000 aviators, with a similar increase in aviation mechanics, armorers, and other technicians. The Navy had developed not only 'aeroplane' aviation, but also dirigibles and seaplanes, and a sizeable number of these wound up in NAS Pensacola storage, in hangars stretching a mile down the Pensacola beach.

Between the wars, Pensacola's activity level dropped considerably, but in 1935 the Navy instituted the cadet training program, and added two more training centers. As world tensions increased in the late 1930s, training rates increased, and Pensacola became a hub of activity, "the Annapolis of the Air," putting aviators in cockpits as fast as they could be trained. This moderated after the war, but with Naval aviation held steady through the start of the Cold War, the Korean War, and Vietnam War. Navy pilots John Glenn and Neil Armstrong trained at NAS Pensacola. The central location and central function of Pensacola led to the relocation of the Blue Angels demonstration team to NAS Pensacola in 1954. In the 1970s further technical training was located at Pensacola, continuing to the present.

In the 2004 the station suffered a direct hit by Hurricane Ivan, suffering damage to every building, with entire sections destroyed. This led to a massive rebuilding project, including new and remodeled housing, base exchange, and other facilities. The base rescue swimming pool was damaged in the hurricane, and when excavation began to replace it, the workers discovered the wreck of a Spanish ship from the 16th Century, which led to an archeological dig. Pensacola was originally the site of a Spanish fort, which was destroyed only weeks after construction in a hurricane in 1559; this ship may have been one of the ships destroyed at that time.

Pensacola remains a vital location for US Naval aviation and training.