Evers served in the U.S. Army in Europe during World War II. Afterward he and his elder brother, Charles Evers, both graduated from Alcorn Agricultural and Mechanical College (now Alcorn State University, Lorman, Miss.) in 1950. They settled in Philadelphia, Miss., and engaged in various business pursuits—Medgar was an insurance salesman, and Charles operated a restaurant, a gas station, and other enterprises—and at the same time began organizing local affiliates of the National Association for the Advancement of Colored People (NAACP) (National Association for the Advancement of Colored People). They worked quietly at first, slowly building a base of support; in 1954 Medgar moved to Jackson to become the NAACP's first field secretary in Mississippi. He traveled throughout the state recruiting members and organizing voter-registration drives and economic boycotts.

During the early 1960s the increased tempo of civil-rights activities in the South created high and constant tensions, and in Mississippi conditions were often at the breaking point. On June 12, 1963, a few hours after President John F. Kennedy had made an extraordinary broadcast to the nation on the subject of civil rights, Medgar Evers was shot and killed in an ambush in front of his home. The murder made Evers, until then a hardworking and effective but relatively obscure figure outside Mississippi, a nationally known figure. He was buried with full military honours in Arlington National Cemetery and awarded the 1963 Spingarn Medal of the NAACP.

Charles Evers immediately requested and was granted appointment by the NAACP to his brother's position in Mississippi, and afterward he became a major political figure in the state. Evers's widow, Myrlie Evers-Williams (Evers-Williams, Myrlie), was the first woman to head the NAACP (1995–98).

Byron de La Beckwith, a white segregationist, was charged with the murder. He was set free in 1964 after two trials resulted in hung juries but was convicted in a third trial held in 1994. Beckwith was given a life sentence, and in 2001 he died in prison.