By June 1940 Duxford was placed in a high state of readiness. 19 Squadron moved to nearby Fowlmere. The dominance of the skies over Britain would be totally crucial to keeping German forces out of the country, this became known as The Battle of Britain. Hurricanes first arrived at Duxford in July with the formation of 310 Squadron, which consisted of Czechoslovakian pilots who had escaped from France. At the end of August Air Vice-Marshal Trafford Leigh-Mallory, ordered the Hurricanes of 242 Squadron commanded by Douglas Bader to come down from RAF Coltishall to join 19 and 310 Squadrons which were on daily standby at Duxford. These units, led by Bader, became known as the "Duxford Wing", the first of 12 Group's "Big Wing" formations.

In recognition of the efforts, achievements and sacrifices made by the squadrons and airmen during the Battle of Britain, the "gate guard" aircraft on display at the entrance gate to IWM Duxford is a Hawker Hurricane II, squadron code WX-E of No.302 (Polish) Squadron, Serial No. P2954, flown by Flight Lieutenant Tadeusz Pawel Chlopik, RAF (Polish Air Force).

Duxford became the home of several specialist units, including the Air Fighting Development Unit (AFDU), which moved to the station at the end of 1940. The AFDU's equipment included captured German aircraft, which were restored to flying condition for evaluation. Duxford was important in developing the Hawker Typhoon into a formidable low-level and ground attack fighter, and also the suggestion of re-engining the Mk1 North American P-51 Mustang with the Merlin. In 1942 the first Typhoon Wing was formed. Its first operation took place on 20 June 1942.

Other RAF Fighter Command units which operated from Duxford were : 19, 56, 66, 133, 181, 195, 222, 242, 264, 266, 310, 312, 601, 609, 611 Squadrons and the AFDU.

Duxford airfield was assigned to the United States Army Air Forces in 1943 .It was allocated to the Eighth Air Force fighter command.

Duxford was the initial home of the 5th Air Defense Wing

The air echelon moved to Oujda, French Morocco during January–February 1943. After this the last RAF units moved out and on 15 June 1943 Duxford was officially handed over to the Eighth Air Force. 78th Fighter Group

The 78th FG was first equipped with P-47s and converted to P-51 Mustangs in December 1944. The group flew many missions to escort Boeing B-17 Flying Fortress and Consolidated B-24 Liberator bombers that attacked industrial complexes, submarine yards and docks, V-weapon sites and other targets in Continental Europe.