The regiment moved to Covington, Kentucky, on September 1, 1862, in preparation for the Defense of Cincinnati against a threatened Confederate invasion by troops under Edmund Kirby Smith.

In August, it moved with General Ambrose E. Burnside's army to East Tennessee where it participated in the capture, occupation, and defense of Knoxville during the fall and early winter.

Following a brutal winter at Strawberry Plains, TN in pursuit of James Longstreet's retreating forces, it was assigned to duty as part of the XXIII Corps for the Atlanta campaign.

The 104th played a key role in the 1st Brigade, 3rd Division's unsuccessful assault on Confederate fortifications at Utoy Creek on August 6, 1864, where it sustained its heaviest casualties of the war up to that point.

The regiment fought a skirmish near Wilmington, NC and was near Raleigh when word came of General Robert E. Lee's surrender at Appomattox, Va. During its service it was assigned to: The 104th OVI mustered out of the army on June 17, 1865.