Although outbreaks of Mycobacterium abscessus infection have been reported, none of these reports has identified the potential sources of infection and modes of transmission. In April 2008, we identified and investigated an outbreak of M. abscessus skin and soft tissue infections following acupuncture among the patients who visited an oriental medical clinic. Active surveillance of patients who had visited the clinic was conducted to define the extent of the outbreak. Environmental cultures and a case–control study were performed to elucidate the source of infection and mode of transmission. From 1002 patients interviewed, 109 patients were identified as having suffered M. abscessus skin and soft tissue infections at acupuncture sites. A single strain of M. abscessus was isolated from the wounds of 31 patients and nine environmental samples, including a diluted glutaraldehyde solution. The case–control study revealed that a higher numbers of visits to the clinic for acupuncture (adjusted OR (aOR) 20.12; 95% CI 4.34–93.35) and the use of interferential current therapy or low-frequency therapy (aOR 36.12; 95% CI 5.54–235.44) were associated with the development of M. abscessus infection. The contaminated diluted glutaraldehyde solution that was used to disinfect the physical therapy devices may have been the source of the outbreak of M. abscessus infection in the 109 patients who underwent acupuncture.