Microbubbles rapidly shrink and collapse on receiving physical stimuli. The impulses or OH free radicals produced by the microbubble collapse are considered to have a bactericidal effect, although the precise mechanism involved remains unknown. Among bactericidal agents, ozone is highly effective in inactivating many microorganisms. In this study, the bactericidal effect of ozone microbubbles on Bacillus subtilis was investigated by changing parameters such as outlet pressure of pump, elapsed time, concentration of ozone fed and dissolved, and microbubble formation methods. By converting ozone to microbubbles, the high bactericidal effect was obtained with a small volume of ozone.