Introduction: Root canal treatment often fails because of bacteria that grow back after obturation, such as Enterococcus faecalis (E. faecalis). The previous study has found the percentage of root canal treatment failure as much as 32-70%. It was because the facultative anaerobic bacteria are able to penetrate into the dentinal tubules and found to be resistant. Ambonese banana stem is one of the herbal plants in Indonesia that has been widely used, including the sap on the stem. Active substances in the sap of Ambonese banana stems such as flavonoids, saponins, and tannins have antimicrobial efficacy. The purpose of this study is to prove the antibacterial activity of Ambonese banana stems extract on E. faecalis and analyzing bacterial viability through a fluorescent microscope. Methods: The sample of this study was E. faecalis (ATCC 29212) which bred on BHIB media, then serially carried out dilution with Ambonese banana stem extract with a composition of 100%, 50%, 25%, 12.5%, 6.25%, 3.125 %, 1,563%. and 0.781%. Cultured in the Agar Mueller-Hinton medium and then calculate the number of bacterial colonies. This examination was continued using a fluorescent microscope for determining the viability of bacteria. Results: We obtained MIC at a concentration of 1.563% and MBC at a concentration of 3.125%. The ability of Ambonese banana stem extracts to kill E. faecalis in MIC was 92.22%, while in MBC it was 100%. Conclusion: Ambonese banana stem extract effectively inhibited the growth of E. faecalis bacteria.