Kitolod flower (Isotoma longiflora (L.) C.Presl.) is one of the native plants of Indonesia which is widespread in various regions in Indonesia. Traditionally, Kitolod flower has been used to treat dental diseases. Kitolod flower contains flavonoids, terpenoids, tannins and alkaloids which can be antibacterial so that it is thought to be able to inhibit the growth of bacteria that cause tooth disease. This study aims to determine the inhibitory test of methanol extract and its fraction on Streptococcus mutans bacteria in vitro. with diffusion method. The results showed that at a concentration of 1%, hexane and ethyl acetate fractions have a moderate inhibition zone response with a clear zone diameter of 17.98 mm and 17.03 mm. Methanol fraction has a weak inhibition zone response with clear zone diameter of 11.66 mm against the growth of Streptococcus mutans. From this study concluded that methanol extract and its fraction have antibacterial activity against Streptococcus mutans bacteria.