Biofilm formation in dental unit water systems (DUWSs) can contaminate water from three-in-one syringes, air rotors, and low-speed handpieces. This may serve as a potential source of infection for dentists, dental staff, and patients, so these systems must be sterilized. Because slightly acidic electrolyzed water (SAEW) is often used as a disinfectant for food, the aim of this study was to investigate the possibility of using SAEW as a DUWS disinfectant. Slightly acidic electrolyzed water was injected into a dental unit and its effects evaluated. Chemical properties such as chlorine ion and potential hydrogen in the SAEW were measured. Detection of both ordinary and heterotrophic bacteria from the DUWS was performed by culture, and biofilm formation of the bacteria in the DUWS evaluated. Polymerase chain reaction (PCR) was used to detected contamination by nosocomial pathogens. Almost all the chlorine ions in the SAEW were exhausted during the two-day trials, and the pH value of the SAEW fell from 5 to 4. No viable cells were detected in the SAEW collected. Biofilm formation in the water from the DUWS with SAEW was almost at a baseline level, whereas that without SAEW was 4 times higher. The PCR analysis showed that no nosocomial infecting pathogens were detected in the SAEW. The present study demonstrated the antiseptic effect of SAEW in DUWS.