Charles graduated from the medical school of Northwestern University in 1888 and joined his father, william Worrall Mayo, and older brother, william James Mayo, in their private medical practice in Rochester, Minnesota.

He was also responsible for the clinic's ophthalmic patients until 1908. He and early partners insisted on sterile conditions in the operating room, and that was one of many factors which contributed to the medical practice's early surgical successes.

The Mayos' first partner was Augustus Stinchfield, who was hired by william Worrall Mayo. Once in place as a partner in the private practice, W. W. Mayo retired at age 73. The private practice became the not-for-profit Mayo Clinic in 1919. At that point, the remaining partners went on salary, and the Mayo Properties Association was established. The world's first "integrated group practice" was established by the seven partners and staff.

Mayo retired in 1930 and died of pneumonia in 1939 in Chicago, Illinois. His two sons Charles william Mayo and Joseph Graham Mayo both worked at the clinic. Joseph Graham Mayo was killed in November 1936 in an accident when a train hit his car killing him and his hunting dog, Floosie. Mr. Mayo and his dog were buried in the same casket. And a grandson, Charles Horace Mayo II, served a residency at the clinic. The United States Postal Service released a stamp on September 11, 1964 depicting Charles Horace Mayo and his brother.