Mayo earned his medical degree at the University of Michigan Medical School in 1883, where he became a founder of Nu Sigma Nu Medical Fraternity. Afterwards he returned to Rochester to practice medicine alongside his father and his brother Charles.

On August 21, 1883, a tornado struck Rochester, killing 29 people and seriously injuring over 55 others. One-third of the town was destroyed, but young Will and his family escaped serious harm. The relief efforts began immediately with a temporary hospital being established at the town’s dance hall. The Mayo brothers were extensively involved in treating the injured who were brought there for help. Mother Alfred Moes and the Sisters of Saint Francis were called in to act as nurses (despite the fact they were trained as teachers and had little if any medical experience).

William J. Mayo married Hattie Marie Damon (1864-1952) in 1884. They had 5 children, two of whom survived infancy. Carrie born in 1887 and Pheobe in 1897 both married physicians at the Mayo clinic.

After the crisis had subsided, Mother Alfred Moes approached William Worrall Mayo about establishing a hospital in Rochester. On September 30, 1889, Saint Mary’s Hospital opened. W.W. Mayo, 70 years old, became the consulting physician and surgeon at the hospital, and his two sons began seeing patients and performing surgery with the assistance of the Sisters of Saint Francis.

William J. Mayo served in World War I at the rank of Colonel (O6), as chief adviser for U.S. Army surgical services in the office of the U.S. Army Surgeon General. His brother Charles was also a Colonel in the U.S. Army Medical Corps and alternated with William as the associate chief adviser for surgical care of U.S. Army soldiers. When President Woodrow Wilson organized the Committee of American Physicians for Medical Preparedness in 1916, William was named its chairman and Charles as one of its members. That committee subsequently became the General Medical Board of the Council for National Defense. William was made a member of its executive committee and Charles was his alternate.

During World War I, the Mayo Clinic continued to be busy. Many draftees were examined and war training classes were conducted for new physicians in the medical corps. William and Charles designed courses to educate military doctors on the latest scientific and surgical developments. The two brothers divided their time between Rochester and their duties in Washington, D.C. so that one of them would always be at the Clinic. This schedule took a toll on their health; Charles contracted pneumonia during one of his stints in Washington, and William developed hepatitis while in Rochester in 1918.

After hostilities ended in November 1918, both of the Mayo brothers were promoted to the rank of Brigadier General (O7) in the U.S. Army Reserve. In addition, they both received the U.S. Distinguished Service Medal for their wartime service to the country.

In September 1931, Mayo and other prominent individuals of the time were invited by The New York Times to make a prediction concerning the world in eighty years time in the future, in 2011, to celebrate the paper’s hundred-and-sixtieth anniversary since its establishment in 1851. Mayo’s prediction was that the life expectancy of developed countries would reach 70 years, compared to less than sixty years in 1931.

He died in July 1939 of gastric carcinoma (stomach cancer) in Rochester, Minnesota. That type of tumor had been a major focus of his surgical practice. Mayo is buried with his family members near his parents and brother at Oakwood Cemetery in Rochester.

The United States Postal Service printed a stamp depicting him and his brother, Charles Horace Mayo, on September 11, 1964.