On the outbreak of the Second World War, it continued to be used for training bomber crews, disbanding on 4 April 1940 when it merged with 76 Squadron to form No. 16 OTU.
On 1 August 1940 it reformed, becoming the first squadron to equip with the new Short Stirling heavy bomber, the first RAF squadron to operate four engined bombers during the Second World War, flying the first bombing raids with the Stirling against oil storage tanks near Rotterdam on the night of 10/11 February 1941.
It flew on the 1000 bomber raids to Cologne, Essen and Bremen in May and June 1942. It was transferred to the Pathfinder Force in October 1942, with the job of finding and marking targets for the Main Force of Bomber Command bombers. It re-equipped with the Avro Lancaster from 11 May 1943, flying its first mission with the Lancaster on 12 July 1943.
It continued in the Pathfinder role until the end of the war in Europe. It flew its last bomber mission on 25 April 1945 against Wangerooge, and dropped food to starving civilians in the Netherlands in May. While it was planned to fly 7 Squadron out to the Far East to join Tiger Force for air attacks against Japan, the war ended before the squadron was due to move.
The squadron carried out 5,060 operational sorties with the loss of 165 aircraft.