The ninth of ten children born to the actor Junius Brutus Booth, he early showed excellent theatrical potential but also exhibited an emotional instability and a driving egocentricity that made it difficult for him to accept his brother Edwin's rise to acclaim as the foremost actor of the day.

After an unsuccessful Baltimore theatrical debut in 1856, John played minor roles in Philadelphia until 1859, when he joined a Shakespearean stock company in Richmond, Virginia. He was widely acclaimed on a tour of the Deep South in 1860 and remained in demand as an actor throughout the Civil War.

A vigorous supporter of the Southern cause, Booth was outspoken in his advocacy of slavery and his hatred of Lincoln. He was a volunteer in the Richmond militia that hanged the abolitionist John Brown (Brown, John) in 1859. By the autumn of 1864 Booth had begun to plan a sensational abduction of President Lincoln. He recruited several coconspirators, and throughout the winter of 1864–65 the group gathered frequently in Washington, D.C., where they mapped out a number of alternative abduction plans. After several attempts had miscarried, Booth resolved to destroy the president and his officers no matter what the cost.

On the morning of April 14, 1865, Booth learned that the president was to attend an evening performance of the comedy Our American Cousin at Ford's Theatre in the capital. Booth hurriedly assembled his band and assigned each member his task, including the murder of Secretary of State William Seward (Seward, William H). He himself would kill Lincoln. About 6:00 PM Booth entered the deserted theatre, where he tampered with the outer door of the presidential box so that it could be jammed shut from the inside. He returned during the play's third act to find Lincoln and his guests unguarded.

Eleven days later, on April 26, Federal troops arrived at a farm in Virginia, just south of the Rappahannock River, where a man said to be Booth was hiding in a tobacco barn. David Herold, another conspirator, was in the barn with Booth. He gave himself up before the barn was set afire, but Booth refused to surrender. After being shot, either by a soldier or by himself, Booth was carried to the porch of the farmhouse, where he subsequently died. The body was identified by a doctor who had operated on Booth the year before, and it was then secretly buried, though four years later it was reinterred. There is no acceptable evidence to support the rumours, current at the time, doubting that the man who had been killed was actually Booth.