NS Mayport is at the mouth of the St. John's River, which has been used by ships going back at least as far as 1522, when the French captain Commodore Jean Ribault landed in this area. What is today the Jacksonville, Florida area was the stage for a largely naval struggle between French and Spanish forces in the middle of the 1500s; the Spanish won, but never made a strong push to settle Florida. During the American War of Independence, the British seized the area and used it to stage naval operations, to prevent American Revolutionaries from basing out of Spanish territory.

The area was not a major theater of operations in the Civil War, and the station was only opened in 1938, as part of the gradual defense buildup before World War Two. There was an effort underway at that time to decentralize the East Coast Navy from its traditional stations in New England. The new stations in Florida had grand mission goals: facilities for up to four carrier groups, six patrol squadrons, a substantial maintenance and berthing center, and an air station. Wartime realities shifted most Navy activity in Florida toward other stations, mainly antisubmarine patrols, but Mayport did become site of a Naval Section Base, including an air station. The air station expanded as the war went on, eventually taking the base over.

After the war Mayport station was deactivated and decommissioned; it became a Coast Guard boot camp until 1947. In 1948, with rising international tension, Mayport was reactivated as an Outlying Field. In the 1950s the base was expanded, and more land acquired. Navy ship traffic started to flow through Mayport. The river channel was dredged, to allow carrier sized craft to dock, and NAAF Mayport gained its first carrier, the USS Franklin D. Roosevelt, in 1955. From that point on, Mayport grew in surface ships stationed. Curiously, the base was not actually designated a Naval Air Station until 1988.

NS Mayport has two unusual roles in communications history. In 1959 Mayport became a link in the Navy's automatic teletype system, the 1950s and 60s version of texting; this led to an immediate 70% increase in communication speed. Later the same year, Mayport received a shipment of mail by missile; the test was mainly for technical reasons, but actual stamped mail was delivered. One of these communications technologies had a future, and one did not.

1962 was a big year for Mayport. In early 1962 ships ported at Mayport recovered astronauts from a Gemini mission. In later 1962, ships from Mayport enforced the Cuban quarantine during the Cuban Missile Crisis. The 1980s saw Mayport ships involved in Operation Urgent Fury, a rescue operation in Grenada. The 1990s were busy at Mayport. Mayport ships were dispatched to Operations Desert Shield and Desert Storm, and supported NATO operations in Yugoslavia. Ships from Mayport have also supported operations in the ongoing war on terror.

The early 2000s saw a base refurbishment, including improved recreation facilities, housing, and overall capability. Currently are six frigates, four destroyers, and four cruisers homeported at NS Mayport, with discussion at brass levels about the carrier future at Mayport.