The regiment was organized in and around Binghamton, New York, and was mustered in for a three-year enlistment on August 27, 1862. It left New York on August 30, 1862, to serve as a guard to the Washington, D.C. railroads in Annapolis Junction, and Laurel, Maryland. On May 4, 1864, the 109th New York served in Virginia. They fought in the Overland Campaign leading up to the Siege of Petersburg, including the battles of The Wilderness, Spotsylvania Courthouse, and Cold Harbor.