O'Neil was born in Carrabelle, Florida. Due to racial segregation, he was denied the opportunity to attend high school in Sarasota, Florida or play in the major leagues. His baseball career began with the Memphis Red Sox in 1937, and he was traded to the Monarchs the following year. A World War II tour in the U.S. Navy from 1943-1945 briefly interrupted his playing career.

O'Neil had a career batting average of .288, including four .300-plus seasons at the plate. In 1946 the first baseman led the league in hitting with a .353 average and followed that in 1947 with a career best .358 mark. He posted averages of .345 and .330 in 1940 and 1949 respectively. In 1948 he took over as manager of the Monarchs and guided them to three league titles in 1951, 1953, and 1955. He played in four East-West All-Star games and two Negro League World Series. O'Neil also joined the legendary Satchel Paige as a teammate during the height of the Negro League barnstorming of the 1930s and 1940s to play countless exhibition games.

O'Neil left the Monarchs following the 1955 season, and in 1956 became a scout for the Chicago Cubs. He was named the first black coach by the Cubs in 1962 and is credited for signing Hall of Fame player Lou Brock to his first pro contract. He is sometimes credited with also having signed Hall of Famer Ernie Banks to his first pro contract, but only signed him to his first MLB contract, as Banks had been scouted and signed to the Monarchs by Cool Papa Bell, manager of the Monarchs' barnstorming "B" team, in 1949. Banks played for the Monarchs in 1950 and briefly in 1953 when O'Neil was his manager.

He has worked as a Kansas City Royals scout since 1988 and was named "Midwest Scout of the Year" in 1998.

O'Neil gained national prominence with his compelling narration of the Negro Leagues as part of Ken Burns' PBS documentary on baseball. Since then he has been the subject of countless national interviews, including appearances on Late Night with David Letterman and the Late, Late Show with Tom Snyder.

Today, Buck O'Neil serves as honorary Board Chairman of the Negro Leagues Baseball Museum (NLBM) in Kansas City, Missouri. He was a member of the 18-member Baseball Hall of Fame Veterans Committee from 1981 to 2000 and was instrumental in the induction of eight Negro League players during that time.

O'Neil was a candidate for Hall of Fame induction in a special vote for Negro League players, managers, and executives in 2006, but he did not receive the necessary nine votes from the 12-member committee. O'Neil commented after hearing that he had not been elected to the Hall at age 94. "God's been good to me," he told about 200 well-wishers who had gathered to celebrate but instead stood hushed and solemn. "They didn't think Buck was good enough to be in the Hall of Fame. That's the way they thought about it and that's the way it is, so we're going to live with that. Now, if I'm a Hall of Famer for you, that's all right with me. Just keep loving old Buck. Don't weep for Buck. No, man, be happy, be thankful."

On May 13, 2006 O'Neil received an honorary doctorate in education from Missouri Western State University, where he gave their commencement speech.