At the outbreak of the Civil War, Birney entered the Union Army on May 22, 1861 as a captain in the 1st New Jersey Infantry Regiment. He first saw combat in the First Battle of Bull Run. Promoted to major of the 4th New Jersey Volunteer Infantry, he participated in the battles of Second Bull Run, Chantilly, Fredericksburg, and, as a colonel, Chancellorsville. In 1863–64, he was appointed as one of three superintendents in charge of enlisting colored troops into the Union army, and in that capacity organized seven regiments. He was named Colonel of the 22nd U.S. Colored Troops. On June 9, 1863, he was commissioned as a brigadier general of volunteers, to rank from May 22, 1863, and assigned to Maryland to recruit more black troops. In 1864, he marched his regiments to fight in South Carolina as a part of the Department of the South. They fared poorly, but did much better work in campaigns in Florida, including the Battle of Olustee.