Many oral microorganisms possess hydrophobic outer surfaces. A two-phase, oil-water mouthwash has, therefore, recently been developed to remove such oral microorganisms. The oil phase consists of olive oil and other essential oils. The aqueous phase includes cetylpyridinium chloride, which is a disinfectant that promotes the adhesion of microorganisms to oil droplets. This study determined the effects of this mouthwash on the production of volatile sulfide in vivo and in vitro. Neither rinsing with water nor brushing teeth decreased the concentration of sulfide in mouth air at 3.5 h after treatment. A reduction of only 30% of sulfide was observed when a commercial mouthwash was used. However, this study demonstrated that use of the two-phase mouthwash led to approximately 80% reduction of sulfide. Furthermore, volatile sulfide and 2-ketobutyrate productions from methionine in a saliva putrefaction system were completely inhibited by the two-phase mouthwash; and consumption of methionine was decreased by 65 percent. It is concluded that the two-phase mouthwash strongly inhibits the production of volatile sulfide.