RAF Mildenhall was built in the late 1920s and early 1930s to meet what British military planners called a "continental threat." It opened as one of the RAF's largest bomber stations on 16 October 1934, with Wing Commander Francis Linnell as its first commander. King George V reviewed 350 aircraft at Mildenhall in 1935 on the occasion of his Silver Jubilee. On 3 September 1939, the day Britain declared war on Germany, three Vickers Wellington aircraft from No. 149 Squadron at Mildenhall were among the first to fly — dispatched to bomb the German naval fleet at Wilhelmshaven. Throughout the war, Mildenhall transitioned from Wellingtons to Short Stirlings to Avro Lancasters. By 1945, aircraft from Mildenhall and its satellite airfields had dropped over 23,000 tons of explosives, laid 2,000 mines, and flown more than 8,000 sorties — at the cost of over 200 bombers and more than 2,000 aircrew. Among those killed was Pilot Officer Rawdon Hume Middleton, an Australian who was posthumously awarded the Victoria Cross.

During the last two weeks of March and the first two weeks of April 1941, Mildenhall was temporarily renamed Millerton Aerodrome and used for the filming of *Target for Tonight*, a Crown Film Unit documentary about the planning and execution of a bombing raid on Germany. The film crew used real Wellingtons and real crews from No. 149 Squadron — with names and identifying details altered so as not to provide intelligence to the enemy. The result was one of the most acclaimed British propaganda films of the war, seen by millions. Mildenhall's role in it went uncredited, for the same reason the base was given a fictional name.

After the war, Mildenhall became a joint RAF-USAF operation in July 1950. Strategic Air Command assigned its first units in 1951, beginning with B-50 Superfortresses. Over the following decades the base's mission shifted from strategic bombing to aerial refueling and reconnaissance. In the late 1970s and through the 1980s, U-2 and SR-71 Blackbird operations ran from Mildenhall — aircraft that flew at altitudes and speeds that made them effectively invisible to adversaries of the era. In June 2021, President Joe Biden and First Lady Jill Biden flew into Mildenhall on Air Force One, en route to the G7 summit in Cornwall, and Biden addressed troops on base shortly after landing. The base today is home to the 100th Air Refueling Wing, flying KC-135R Stratotankers, and the 352nd Special Operations Wing, flying CV-22 Ospreys and MC-130J Commando IIs.

In January 2015, the US Department of Defense announced that operations at Mildenhall would be relocated to Germany as part of a European cost-cutting consolidation. The announcement set off years of uncertainty. By 2017, the closure was on indefinite hold. In July 2020, the decision was reversed: the 100th Air Refueling Wing and 352nd Special Operations Wing would remain at Mildenhall indefinitely. The base — alongside its sister installation RAF Lakenheath — continues to anchor the largest US Air Force presence in the United Kingdom. In late 2024, unidentified drones were spotted over Mildenhall and other US air bases in East Anglia, prompting a security response that included the deployment of RAF Regiment personnel with counter-drone systems.