Opdycke enlisted immediately following the First Battle of Bull Run and was commissioned a first lieutenant in the 41st Ohio Infantry on August 26, 1861. As a captain, he fought in the Battle of Shiloh. He resigned in September 1862 to help recruit the 125th Ohio Infantry, in which he became the lieutenant colonel on October 1, 1862, and the colonel on January 14, 1863. His regiment earned fame in the defense of Horseshoe Ridge at the Battle of Chickamauga. In command of a demi-brigade, Opdycke's men were some of the first to reach the summit of Missionary Ridge during the Battles for Chattanooga. He fought throughout the Atlanta Campaign with the Army of the Cumberland and was badly wounded at the Battle of Resaca, but he recovered sufficiently to lead an assault in the Battle of Kennesaw Mountain.