Background: Allergic to iodine antiseptic material was found at several hospital, one of them from National Taiwan University Hospital showed 34% patient allergic to iodine as antiseptic. Staphylococcus aureus is an anaerobic gram positive bacteria as ethiology of oral cavity infections. Infections in the oral cavity can be prevented by an antiseptic . Kaffir lime fruit (Citrus hystrix) contains flavonoid, saponin, tannin and essential oils have an inhibitory effect to the bacteria by destroying and inhibit the formation of the bacterial cell wall. Objective: This research aims to determine the effectivity of 96% Citrus hystrix extract etanol at several concentration to inhibit the growth of Staphylococcus aureus. Methods: The method of this research is in vitro experimental laboratory. Results: The result showed that Citrus hystrix extract etanol 96% at concentration 100% is the most effective concentration to inhibit the growth of Staphylococcus aureus. Conclusion: It was concluded that the Citrus hystrix extract can inhibit the growth of Staphylococcus aureus and have a potential as a candidate of antiseptic in the iodine allergy case.