In April 1861, at the outbreak of the American Civil War, Wikoff enlisted as a private in the 1st Pennsylvania Infantry. The next month he was commissioned a first lieutenant in the 15th U.S. Infantry. He was shot in the left eye at the Battle of Shiloh and wore an eye patch throughout the rest of his life. He also participated in the Battle of Chickamauga and the Battle of Missionary Ridge, for which he was a brevetted major. He was promoted to captain in August 1864.