Objective To understand the transmission mode of patients with different sites of Clostridium perfringens (C. perfringens) infection as well as prevention and control measures of healthcare-associated infection (HAI). Methods Clinical information, HAI prevention and control measures and environmental sampling results of three patients with different sites of C. perfringens infection in a hospital were retrospectively collected, and rela-ted literature was reviewed for analysis and summary. Results Three cases of C. perfringens infection were gas gangrene of pelvic cavity, liver abscess, and bloodstream infection. C. perfringens was isolated from air-returning outlet (fence + filter screen) of negative pressure laminar flow operating room and negative pressure laminar flow ward of a patient, environmental hygiene detection results of the other 2 cases were normal. Conclusion It is necessary to strictly carry out environmental cleaning, disinfection and isolation for patients infected with C. perfringens, long distance dissemination of C. perfringens from infected sites of patients with gas gangrene can be produced, it is recommended to use independent non-laminar flow operating room, patients with infection at other sites can be isolated in a single room or shared room with patients of the same disease.