The Ro.41 found a role as a trainer aircraft, for which it was well-suited, and a series of 30 two-seat aircraft first flew in 1937. The Ro.41 replaced the Breda Ba.25, and soon another 264 single-seat and 66 two-seat models were ordered.
The Ro.41 was also proposed as light fighter. Twenty-eight were sent to Spain where, thanks to their high rate of climb, they acted as point-defence interceptors around Seville, though it appears that they did not score any victories.
It served in 5 and 50 Wing as a fighter bomber, before the Breda Ba.65 arrived. XVI Gruppo, 50 Stormo, had all its three squadrons equipped with Ro.41s. 163 Sqn was sent to Rodi and used the Ro.41 as a fighter until 1940. Twelve Ro.41s served with 160 Gruppo in 1939 and were used as first line fighters, though the Gruppo was based on CR.32s and CR.42s. From 10 August 1940 four Ro.41s of 159 Sqn, 12 Gruppo, 50 Assault Wing were flown from Tobruk as night fighters.
In its limited career as first line fighter the Ro.41 did not achieve any victories, and it is unlikely that it was ever involved in any air-combats. By this time even the CR.32 and CR.42s were obsolete, and the Ro.41s were only a stop-gap measure. Their real task was advanced training and despite the obsolete design they managed to be popular, reliable and cheap machines. They were also built by Agusta and AVIS.
The Ro.41bis, with a smaller wing and better performance was tested, but the CR.32 was already available for flying schools, and it was not a success. In September 1938 MM.3786 was sent to Uruguay to display the type, but no orders were placed.
Ro.41s were popular aircraft and for many years first line squadrons and flight schools operated it, until it became obsolete for first line use. One of the few changes was the fitting of the Piaggio P.VII RC.35 engine, that had a single-stage compressor which gave 500 hp at low level. Guns were seldom fitted, and two-seat versions had no weapons at all, and also carried less fuel.
Production numbered 726 aircraft by 1943. After the armistice the RSI's Aeronautica Nazionale Repubblicana operated some aircraft, and the Luftwaffe used them as trainers in Germany and France. Strangely no examples remained in Southern Italy, perhaps because flight schools, like Castiglione del Lago airfield, were in the central and northern Italy. Five ANR aircraft survived the war.
The Ro.41 was the first post-war aircraft to enter production when an order was sent to Agusta for 15 new aircraft (5 single and 10 two-seaters) and later ten more (7 single and 3 two-seaters). These aircraft had a wooden propeller, possibly spare parts still in store. They were painted silver, the new standard for Italian aircraft, instead of camouflage colours. Three of these machines formed the first acrobatic team of the Aeronautica Militare in 1947 at Padua. These aircraft were flown until 1950.
In total production reached 753 aircraft.