Booth made his stage debut at age 17 on August 14, 1855 in the supporting role of the Earl of Richmond in Richard III at Baltimore's Charles Street Theatre. The audience jeered at the inexperienced actor when he missed some of his lines. He also began acting at Baltimore's Holliday Street Theater, owned by John T. Ford, where the Booths had performed frequently. In 1857, Booth joined the stock company of the Arch Street Theatre in Philadelphia, Pennsylvania where he played for a full season. At his request, he was billed as "J.B. Wilkes", a pseudonym meant to avoid comparison with other members of his famous thespian family. Jim Bishop wrote that Booth "developed into an outrageous scene stealer, but he played his parts with such heightened enthusiasm that the audiences idolized him." In February 1858, he played in Lucrezia Borgia at the Arch Street Theatre. On opening night, he experienced stage fright and stumbled over his line. Instead of introducing himself by saying, "Madame, I am Petruchio Pandolfo", he stammered, "Madame, I am Pondolfio PetPedolfio PatPantuchio Peddammit! Who am I?", causing the audience to roar with laughter.
Later that year, Booth played the part of Mohegan Indian Chief Uncas in a play staged in Petersburg, Virginia, and then became a stock company actor at the Richmond Theatre in Virginia, where he became increasingly popular with audiences for his energetic performances. On October 5, 1858, Booth played the part of Horatio in Hamlet, alongside his older brother Edwin in the title role. Afterward, Edwin led the younger Booth to the theatre's footlights and said to the audience, "I think he's done well, don't you?" In response, the audience applauded loudly and cried, "Yes! Yes!" In all, Booth performed in 83 plays in 1858. Among them were William Wallace and Brutus, having as their theme the killing or overthrow of an unjust ruler. Booth said that, of all Shakespearean characters, his favorite role was Brutus, the slayer of a tyrant.
Some critics called Booth "the handsomest man in America" and a "natural genius", and noted his having an "astonishing memory"; others were mixed in their estimation of his acting. He stood 5 feet 8 inches (1.73 m) tall, had jet-black hair, and was lean and athletic. Noted Civil War reporter George Alfred Townsend described him as a "muscular, perfect man" with "curling hair, like a Corinthian capital".
Booth's stage performances were often characterized by his contemporaries as acrobatic and intensely physical, with him leaping upon the stage and gesturing with passion. He was an excellent swordsman, although a fellow actor once recalled that Booth occasionally cut himself with his own sword.
Historian Benjamin Platt Thomas wrote that Booth "won celebrity with theater-goers by his romantic personal attraction", but that he was "too impatient for hard study" and his "brilliant talents had failed of full development." Author Gene Smith wrote that Booth's acting may not have been as precise as his brother Edwin's, but his strikingly handsome appearance enthralled women. As the 1850s drew to a close, Booth was becoming wealthy as an actor, earning $20,000 a year (equivalent to about $533,000 today).