The raid on the Shenandoah Valley town of Harpers Ferry, Virginia, by the radical abolitionist John Brown in October 1859 was not only a marker of the first hostilities of the Civil War, but also of those against the Baltimore and Ohio Railroad. An express train manned by A. J. Phelps was stopped at Harpers Ferry by Brown and his approximately 150 men. They gained control of the Harpers Ferry bridge and the local United States armory. During the seizure of the train, shots were exchanged and an African American porter was fatally wounded before the rebellion was quelled.

A year and a half later on April 18, 1861, the Baltimore and Ohio Railroad was once again severed at Harpers Ferry. U.S. soldiers burned the surrounding buildings and the rifle factory before they withdrew. Later that evening the Virginia State Militia took control over the area and salvaged the remains. The Virginian soldiers who participated in the events formed the foundation of the Confederate Army of Northern Virginia. This event divided the Baltimore and Ohio Railroad much like the nation as a whole had been divided.

Throughout the war the Baltimore and Ohio Railroad suffered periods in which it was attacked and its service was stalled. In 1861, Confederate colonel Thomas J. Jackson attacked the portion of the Baltimore and Ohio Railroad that crossed the Valley of Virginia. He devastated the line, sequestering locomotives, burning freight cars, and destroying rails. The historian James I. Robertson, however, has argued that accounts of Jackson's raid are "totally fictional" and rely too heavily on unreliable sources.

Throughout 1862 and 1863 the railroad experienced a number of lines closed from local skirmishes. A more notable closure was in 1864 when the Confederacy sought to end the transportation of Union soldiers over the Baltimore and Ohio's western route to Washington, D.C. The mayor of Charles Town, West Virginia, threatened the railroad, stating that if Union soldiers or supplies continued to use the route, then it would be destroyed. While the Baltimore and Ohio Railroad refused to halt Union movements, it suffered severe repercussions when the Virginia State militia destroyed the bridge at Harpers Ferry.

Throughout the war the Baltimore and Ohio Railroad changed hands repeatedly, creating an unstable and destructive environment for the company. The war damaged one of the nation's great railways, creating an inability to provide continuous service to its patrons until after the Civil War. In the postwar period, however, the Baltimore and Ohio Railroad relished in a golden age of growth and prosperity as lines expanded west to Chicago and St. Louis, and advances in railroad technology enabled greater speed and safety for trains.