Mayo spent a brief period of time in Buffalo, New York before settling in Lafayette, Indiana where he worked as a tailor (one of the jobs he had practiced while in England). He returned to medicine in 1849, assisting in a cholera outbreak and then attending courses at Indiana Medical College in La Porte, Indiana where he received his medical degree. While the training there would be considered poor by modern standards, the school did have a microscope, an uncommon tool at the time. Knowledge of this instrument proved to be useful in Mayo's future practice. He received his degree on February 14, 1850.
He brought his family to a village named Cronan's Precinct (near Le Sueur) along the Minnesota River where he became known as the "Little Doctor" because of his 5-foot, 4-inch (163 cm) stature. Mayo tried his hand at a number of different activities including farming, operating a ferry service, and serving as a judge in addition to occasional medical duties. By this time, he had two more daughters in his family, Phoebe and Sarah.
After a flood in 1859, the family moved to a home on Main Street in Le Sueur. There, he set up his first official medical practice, but the flow of patients was too low to support the family. Mayo took to publishing a shortlived newspaper, the Le Sueur Courier, though it only lasted about three months. He also spent time working on a steamboat. The family saw its first male addition, William James Mayo, in 1861.
As the American Civil War began that same year, Mayo attempted to get a job as a military surgeon, but was rejected. Nonetheless, he soon found his way into military medicine as the Dakota War of 1862 erupted in southwest Minnesota in late 1862. Organizing a group of people from Le Sueur and St. Peter, he headed out to New Ulm, where some of the worst fighting had occurred. Makeshift hospitals in the city cared for people injured in the conflict and refugees driven by fear from farms in the area. His wife opened her home and a nearby barn to host eleven refugee families back in Le Sueur.
William W. Mayo opened a medical practice in Rochester, also spending time as a city mayor, alderman, and member of the school board. Here, the number of patients was large enough to support the family, so he never had to take on additional jobs again. He spent some time in New York and Pennsylvania in 1869 studying surgical techniques, although the doctor had become quite successful on his own.
The event where the Mayo Clinic story usually begins happened 14 years later in 1883, when a tornado devastated Rochester. With the assistance of his sons, other doctors who came to help, and the local Sisters of Saint Francis of Rochester, Minnesota, he organized treatment of the injured. Mother Alfred Moes of the Sisters of St. Francis convinced him to help her establish a a new hospital under her direction, forming St. Marys Hospital in 1889. At the time, only three people were on the surgical staff: William Worrall Mayo as chief, and his two sons as the medical practitioners (their father was 70 by this time). No other doctors accepted invitations to join at the time, perhaps because St. Marys was a Catholic Hospital. The alliance between the Episcopalian Mayos and the Roman Catholic Franciscan religious order caused some controversy at the time.
Dr. W. W. Mayo died in 1911, with his wife passing away four years later. They are buried next to each other at Oakwood Cemetery in Rochester.
The family's home in Le Sueur was added to the National Register of Historic Places in 1969. Carson Nesbit Cosgrove and his family later lived in the home. Cosgrove went on to help create the Minnesota Valley Canning Company, later named Green Giant. His son and grandson also headed the company in later years.