The first Bf 109As served in the Spanish Civil War. By September 1939, the Bf 109 had become the main fighter of the Luftwaffe, replacing the biplane fighters, and was instrumental in gaining air superiority for the Wehrmacht during the early stages of the war. During the Battle of Britain, it was pressed into the role of escort fighter, a role for which it was not originally designed, and it was widely employed as a fighter-bomber, as well as a photo-reconnaissance platform. Despite mixed results over Britain, with the introduction of the improved Bf 109F in early 1941, the type again proved to be an effective fighter during the Invasion of Yugoslavia (where it was used by both sides), the Battle of Crete, Operation Barbarossa (the invasion of the USSR) and the Siege of Malta.

In 1942, it began to be partially replaced in Western Europe by a new German fighter, the Focke-Wulf Fw 190, but it continued to serve in a multitude of roles on the Eastern Front and in the Defense of the Reich, as well as in the Mediterranean Theatre of Operations and with Erwin Rommel's Afrikakorps. It was also supplied to several of Germany's allies, including Italy, Finland, Hungary, Romania, Bulgaria, Croatia and Slovakia.

More aerial kills were made with the Bf 109 than any other aircraft of World War II.[77] Many of the aerial victories were accomplished against poorly trained and badly organized Soviet forces in 1941 during Operation Barbarossa. The Soviets lost 21,200 aircraft at this time, about half to combat.[78] If shot down, the Luftwaffe pilots might land or parachute to friendly territory and return to fight again. Later in the war, when Allied victories began to bring the fight closer, and then in German territory, bombing raids supplied plenty of targets for the Luftwaffe. This unique combination of events — until a major change in American fighter tactics occurred very early in 1944, that steadily gave the Allies daylight air supremacy over the Reich — led to the highest-ever individual pilot victory scores.[79] One hundred and five Bf 109 pilots were each credited with the destruction of 100 or more enemy aircraft.[nb 12] Thirteen of these men scored more than 200 kills, while two scored more than 300. Altogether, this group of pilots was credited with a total of nearly 15,000 kills.[77] Though no official "ace" status existed in the Luftwaffe - the term Experte (expert) was used for an experienced pilot irrespective of his number of kills - using the Allied definition of pilots who scored five or more kills, more than 2,500 Luftwaffe fighter pilots were considered aces in World War II.[80] Against the Soviets, Finnish-flown Bf 109Gs claimed a victory ratio of 25:1.[81]

Bf 109s remained in foreign service for many years after World War II. The Swiss used their Bf 109Gs well into the 1950s. The Finnish Air Force did not retire their Bf 109Gs until March 1954. Romania used its Bf 109s until 1955. The Spanish Hispanos flew even longer. Some were still in service in the late 1960s. They appeared in films (notably Battle of Britain) playing the role of Bf 109Es. Some Hispano airframes were sold to museums, which rebuilt them as Bf 109