Acid-electrolyzed functional water (FW) is an efficient bactericide and gargling with FW might be an effective method of oral care. We investigated the possible use of FW as a mouth wash by an in vitro study. The bactericidal effect of FW against different species of bacteria (Staphylococcus aureus, Streptococcus pneumonia, Pseudomonas aeruginosa, and Candida albicans) was evaluated using the numbers of colony-forming units (CFU). The experiment was conducted using PBS, LISTERINE, and ConCool F (undiluted, and the optimal concentration indicated). To investigate the bactericidal mechanism of FW, the activity of superoxide dismutase (SOD), an indicator of oxidative action, was measured in S. aureus. FW was diluted with purified water to concentrations of 10, 30, 50, and 70%. The numbers of CFU were measured for each concentration. XTT assays were performed using HSC-3 and HeLa cells, to examine the viability of the cells following treatment with FW. The same experiment was conducted with PBS, LISTERINE, and undiluted ConCool F. No bacteria treated with FW formed colonies. SOD activity peaked at a 50% concentration of FW and was more than twice that of the control. A significant decrease in the number of CFU was observed following 50% treatment. Since the peaks of the SOD activity and the starting concentrations of the bactericidal effects coincided, the bactericidal effect of FW might be related to its oxidative effects. Bacteria treated with FW had the same survival rate as the other mouth washes. FW might be clinically applicable as a mouth wash.