On the morning of Friday, April 14, 1865, Booth dropped by Ford's Theatre and learned that the president and General Grant were planning to attend the evening performance of Our American Cousin. He held one final meeting with his co-conspirators. He said he would kill Lincoln at the theater (he had since learned that Grant had left town). Atzerodt was to kill Vice-President Andrew Johnson at the Kirkwood House where Johnson resided. Powell was assigned to kill Secretary of State William Seward. Herold would accompany Powell. All attacks were to take place simultaneously at approximately 10:15 P.M. that night. Booth hoped the resulting chaos and weakness in the government would lead to a comeback for the South.

The presidential party arrived at Ford's at about 8:30 P.M. Armed with a single shot derringer and a hunting knife, Booth arrived at Ford's at about 9:30 P.M. Joseph Burroughs, a boy who worked at the theater, held his horse in the rear alley. Booth went next door to a saloon for a drink. He entered the front of Ford's Theatre around 10:07 P.M. Slowly he made his way toward the State Box where the Lincolns were sitting with Clara Harris and Major Henry Rathbone. Lincoln's bodyguard, John Parker of the Metropolitan Police Force, had left his post. At about 10:15 P.M. Booth opened the door to the State Box, shot Lincoln in the back of the head at near point-blank range, and struggled with Rathbone. Booth stabbed Rathbone in the arm and jumped over 11 feet to the stage below. When he hit the floor he snapped the fibula bone in his left leg just above the ankle. Many in the theater thought he yelled "Sic Semper Tyrannis" (Latin for "As Always to Tyrants"). Mrs. Lincoln screamed, Booth flashed his knife at the audience, and he made his way across the stage in front of more than 1,000 people. Everything happened so fast that no one had time to stop him. Booth went out the back door, climbed on his horse, and escaped from the city using the Navy Yard Bridge.