SAS – 1941 and 1942
From November 1941 through to the end of 1942 during the Western Desert Campaign, Mayne participated in numerous night raids deep behind enemy lines in the deserts of Egypt and Libya, where the SAS wrought havoc by destroying many enemy aircraft on the ground. Mayne pioneered the use of military jeeps to conduct surprise hit-and-run raids, particularly on Axis airfields. The National Army Museum stated that Mayne had "a personal tally of more than 100 aircraft destroyed."

The first successful raid at Wadi Tamet in Libya on 14 December 1941, where aircraft and petrol dumps were destroyed, helped keep the SAS in existence, following the failure of the previous initial raid behind enemy lines at Sirte. For his part in the Tamet raid Mayne was awarded the Distinguished Service Order (DSO). Promoted to lieutenant after the second raid of Tamet on 27 December 1941, Mayne also received a mention in despatches on 24 February 1942.

Mayne's official report on the Tamet raid notes:

The following damage was done on or in the vicinity of the aerodrome:
(a) Bombs were placed on 14 aircraft;
(b) 10 aircraft were damaged by having instrument panels destroyed;
(c) Bomb and petrol dumps were blown up;
(d) Reconnaissance was made down to the seafront but only empty huts were found;
(e) Several telephone poles were blown up;
(f) Some Italians were followed, and the hut they came out of was attacked by sub-machine gun and pistol fire and bombs were placed on and around it. There appeared to be roughly thirty inhabitants. Damage inflicted unknown.

Mayne took part in the most successful SAS raid of the Desert War when, on the night of 26 July 1942, with eighteen armed jeeps, British and French commandos raided the Sidi Haneish Airfield. Avoiding detection, they destroyed up to 40 German aircraft escaping with the loss of only three jeeps and two men killed in action.

Commanding Officer
Following Stirling's capture in January 1943, 1st SAS Regiment was reorganised into two separate parts, the Special Raiding Squadron (SRS) and the Special Boat Section (the forerunner of the Special Boat Service). As a major, Mayne was appointed to command the Special Raiding Squadron and led the unit in Sicily and Italy until the end of 1943. In Sicily, Mayne was awarded a bar to his DSO. The official citation reads as follows:

On 10 July 1943, Major Mayne carried out two successful operations, the first the capture of CD battery the outcome of which was vital to the safe landing of 13 Corps. By nightfall SRS had captured three additional batteries, 450 prisoners, as well as killing 200 to 300 Italians. The second operation was to capture and hold of the town of Augusta. The landing was carried out in daylight – a most hazardous combined operation. By the audacity displayed, the Italians were forced from their positions and masses of stores and equipment were saved from enemy demolition. In both these operations it was Major Mayne's courage, determination and superb leadership which proved the key to success. He personally led his men from landing craft in the face of heavy machine-gun fire. By this action, he succeeded in forcing his way to ground where it was possible to form up and sum up the enemy's defences.

In January 1944 Mayne was promoted to lieutenant-colonel and appointed commanding officer of the re-formed 1st SAS Regiment. He subsequently led the SAS with great distinction and valour through the final campaigns of the war in France, the Netherlands, Belgium, Germany and Norway, often campaigning alongside local resistance fighters including the French Maquis. In recognition of his leadership and personal disregard for danger while in France, where he trained and worked closely with the French Resistance, Mayne received a second bar to his DSO.

Lt-Col. R.B. Mayne DSO has commanded 1 SAS Regiment throughout the period of operations in France. On 8 August 1944, he was dropped to Operation Houndsworth base, located west of Dijon, in order to co-ordinate and take charge of the available detachments of his Regiment and co-ordinate their activities with a major Airborne landing which was then envisaged near Paris. He then proceeded in a jeep in daylight to motor to the GAIN base making the complete journey in one day. On the approach of Allied Forces, he passed through the lines in his jeep to contact the American Forces and to lead back through the lines his detachment of twenty jeeps landed for Operation Wallace. During the next few weeks, he successfully penetrated the German and American lines on four occasions in order to lead parties of reinforcements. It was entirely due to Lt-Col. Mayne's fine leadership and example, and his utter disregard for danger, that the unit was able to achieve such striking successes.

During the course of the War, Mayne became one of the British Army's most highly decorated soldiers receiving the DSO with three bars.