Colquitt was born in Monroe, Georgia. His father, Walter T. Colquitt was a United States Representative and Senator from Georgia. Alfred graduated from Princeton College in 1844, studied law and passed his bar examination in 1846. He began practicing law in Monroe. During the Mexican-American War, he served in the U.S. Army at the rank of major. After the war, Colquitt was elected as a member of the United States House of Representatives from 1853 to 1855. He then served in the Georgia state legislature. In 1861, he was a delegate to the state secession convention.

At the beginning of the Civil War, he was appointed captain in the Sixth Georgia Infantry. He saw action in the Peninsula Campaign and the Seven Days' Battles. He rose through the ranks to become a brigadier general in 1862. He led his brigade under Stonewall Jackson in the Battle of South Mountain, Battle of Antietam, the Battle of Fredericksburg, and the Battle of Chancellorsville. After Chancellorsville, some questions arose about Colquitt's abilities, and his brigade was transferred to North Carolina. They were transferred again in the summer of 1863 to protect Charleston, South Carolina. In February of 1864, Colquitt marched his brigade south to help defend against the Union invasion of Florida, and was victorious in the Battle of Olustee. After this battle, Colquitt's brigade rejoined Robert E. Lee's Army of Virginia. Late in the war, the brigade returned to defend North Carolina where Colquitt surrendered in 1865.

He defeated Republican candidate Jonathan Norcross for Governor of Georgia in 1876 and was reelected in 1880 to serve two years under the new state constitution. He was opposed to Reconstruction. In 1883, he was elected as a Democrat to the United States Senate. He was re-elected in 1888 and served until his death in Washington, D.C.