In 1860 O'Sullivan was employed at the studio of Mathew Brady in Washington, D.C. He left in 1862 to work for Alexander Gardner, who published many of his photographs of Civil War battlefields in Gardner's Photographic Sketch Book of the War (1866). In 1867-1869 he joined Clarence King on his expedition along the 40th parallel as official photographer for the U.S. Geological Survey. He photographed with the Darien Survey in Panama in 1870. In 1871-1875 he worked on the Wheeler expeditions to California, Nevada, and Arizona. He documented the culture and landscape of the Southwest Indians in 1874, and in 1880 he was named chief photographer for the Department of the Treasury.