The building began its life in 1833 as the second meeting house of the First Baptist Church of Washington, with Obadiah Bruen Brown as pastor. For nearly three decades it served as a house of worship. Then, in 1861, after the congregation moved to a new structure, theatrical impresario John T. Ford purchased the former church and transformed it into a performance venue he called Ford's Athenaeum. Fire destroyed the building in 1862, but Ford rebuilt it as a proper theater, and it reopened in 1863 to enthusiastic audiences in a capital city desperate for diversion during the Civil War. Within two years, an event inside its walls would make the building's name synonymous with the darkest night in American presidential history.

On the evening of April 14, 1865, just five days after General Robert E. Lee's surrender at Appomattox Court House, President Abraham Lincoln and his wife Mary attended a performance of Tom Taylor's comedy Our American Cousin. The theater held roughly 1,700 people that night. John Wilkes Booth, a famous actor and Confederate sympathizer, entered the presidential box and shot Lincoln in the back of the head with a derringer. Booth stabbed Major Henry Rathbone, who tried to stop him, then leapt from the box to the stage and escaped through the back of the theater. Soldiers carried the unconscious president across the street to the home of tailor William Petersen, where they laid Lincoln on a bed diagonally because of his unusual height. He never regained consciousness, dying at 7:22 the next morning. The theater was immediately shuttered for the criminal investigation. When Ford announced plans to reopen just months later, Secretary of War Edwin Stanton seized the building and an order was issued forever prohibiting its use as a place of public amusement.

What followed the assassination was a long, strange purgatory. The U.S. military took over the building, using it as a warehouse and office complex for the War Department. Records filled the first floor, the Library of the Surgeon General's Office occupied the second, and the Army Medical Museum claimed the third. Then on June 9, 1893, the front section of all three interior floors collapsed when a supporting pillar was undermined during cellar excavation. Twenty-two government clerks were killed and sixty-eight more were injured. Washingtonians whispered that the former church turned theater turned storeroom was cursed. The building was repaired and the clerks returned, but the collapse cemented Ford's Theatre in the public imagination as a place where tragedy seemed to accumulate. A Lincoln museum finally opened on the first floor on February 12, 1932, Lincoln's 123rd birthday. The National Park Service took over the following year.

The restoration of Ford's Theatre was a two-decade campaign driven by an unlikely bipartisan pair: Democratic National Committeeman Melvin D. Hildreth and Republican North Dakota Representative Milton Young. Hildreth first proposed the idea in 1945. A decade of lobbying produced a bill in 1955 for an engineering study. Congress finally approved restoration funds in 1964, and the work was completed in 1968. Vice President Hubert Humphrey dedicated the restored theater on January 21, 1968, and performances resumed ten days later. The theater was renovated again in the 2000s, reopening on February 11, 2009, to commemorate Lincoln's 200th birthday, with President Barack Obama delivering remarks. Today Ford's Theatre seats 665 and produces a full season of plays. The museum beneath the stage holds the derringer Booth used, his diary, the original door to Lincoln's theater box, and the blood-stained pillow from the president's deathbed. Across the street, the Petersen House is furnished exactly as it was the morning Lincoln died there, the federal government's first-ever purchase of a historic home. Together, the theater and the house form Ford's Theatre National Historic Site, a place where American history is simultaneously preserved and performed.