The four prototypes and first production batch of six aircraft were built at Cricklewood, with the first aircraft delivered by road to Hendon on 9 December 1915. The first flight of the first prototype, serial number 1455, was made at Hendon on 17 December, when a short straight flight was made, the aircraft taking off without trouble at 50 mph (80 km/h). A second flight was made the following day, when it was found that the aircraft would not fly faster than about 55 mph (89 km/h). This was blamed on the drag caused by large honeycomb radiators, which were changed to tube radiators mounted on either side of the engine nacelles. A third flight on 31 December revealed a number of control problems: ailerons and elevators were effective but heavy, partly due to excessive friction in the control circuit and the rudders were seriously overbalanced. After minor modifications, the aircraft was flown to RNAS Eastchurch, where full-speed trials were made. On reaching 70 mph (110 km/h), the tail unit began to vibrate and twist violently: the pilot immediately landed and an inspection showed severe damage to the rear fuselage structure but reinforcement failed to cure the problem. The enclosed cockpit and most of the armour plating were also removed. The second prototype, 1456, was completed in April 1916 and had an open cockpit in a longer nose, with room for a gunner's position at the end. To save weight, most of the armour plating was deleted. This was the arrangement for later production of the machine.
After a series of proving flights at Hendon, 1456 was accepted by the RNAS and was flown to Manston for further trials. These revealed that despite a reduced balance area on the elevators, there was still a tail oscillation problem. A lack of directional stability caused by the increased forward side area was partly cured by adding a fixed fin but to find the cause of the tail oscillation, the Admiralty called in F.W. Lanchester from the National Physics Laboratory. He agreed that simple structural weakness was not the root of the problem and that resonance of the fuselage was the probable cause. Static tests on a third prototype, 1457, which had a redesigned, stiffer, fuselage structure showed nothing. This aircraft had an amidships crew position and on 26 June, Lanchester was flown as an observer. The tail oscillations started at 80 mph (130 km/h) and Lanchester observed that the tail was twisting by 15° to either side and deduced that the cause was asymmetric movement of the right and left halves of the elevators, which were not rigidly linked but connected by long control cables. He recommended that the halves of the elevators be connected, removal of the elevator balances and additional bracing between the lower longerons and the lower tailplane spar, measures which were wholly successful.
The fourth prototype, 1458, was completed with the same fuselage structure as 1456 and provision for armament, with a Scarff ring mounting in the nose, a pair of post mountings in the mid position and a gun mounting in the rear fuselage. This was also the first 0/100 to be fitted with uprated 320 hp (240 kW) Eagle engines. After completing acceptance trials, 1456 and 1457 were retained at Manston to form a Handley Page training flight. The first prototype was rebuilt to production standard and 1458 used to test a new nacelle design. This was unarmoured, had an enlarged fuel tank and the fairing was shortened, eliminating the need for the tip to fold. This nacelle design was used on all aircraft built after the initial batch of twelve. From 1461, an additional 130 imp gal (590 L) fuel tank was fitted in the fuselage above the bomb floor. A total of 46 O/100 aircraft were built before being superseded by the Type O/400.
The most significant difference between the two types was the use of 360 horsepower (270 kW) Eagle VIII engines. Unlike the earlier version, this engine was not built in right-handed and left-handed versions, because production of engines of both types for engine type approval had been difficult: wind tunnel tests at the NPL established that the counter-rotating propellers were a cause of the O/100's directional instability, and so it was realised that only one version was necessary, simplifying production and maintenance; the torque effect was overcome by offsetting the fin slightly. The O/400 had a strengthened fuselage, an increased bomb load and redesigned tankage: the nacelle tanks were deleted and fuel was carried in two 130 imp gal (590 L) fuselage tanks supplying a pair of 15 imp gal (68 L) gravity tanks. Deletion of the nacelle tanks permitted a smaller nacelle and simplified supporting struts, the reduction of drag producing an improvement in maximum speed and altitude. The revised nacelle was tested in 3188, which in 1917 was flown at Martlesham Heath with a variety of engine installations. An initial order for 100 of the revised design, to be powered either by Sunbeam Maoris or Eagles, was placed on 14 August but cancelled shortly afterwards. Twelve sets of Cricklewood-built components were transferred to the Royal Aircraft Factory, where they were assembled into the first production O/400s. More than 400 were supplied before the Armistice. Another 107 were licence-built in the USA by the Standard Aircraft Corporation (out of 1,500 ordered by the air corps). Forty-six out of an order for fifty were built by Clayton & Shuttleworth in Lincoln.