Background: Root canal treatment is a dental treatment to maintain vital and non-vital teeth. One of the stages in root canal treatment is irrigation to clean the root canal from necrotic pulp tissue, dentine debris, bacteria, and bacterial products. E. faecalis is one of the bacteria commonly found in infected root canal. Sarang semut extract contain some antibacterial compounds. Objective: To determine the antibacterial activity of sarang semut extract against E. faecalis as an alternative to root canal irrigation materials. Method: This research includes laboratory experimental study in vitro. Sarang semut extract diluted to a concentration of 10%, 20% and 40%, and a solution of 2,5% sodium hypochlorite as a positive control were dropped into agar wells in a petri dish containing agar medium and bacteria. Each test solution was done in six times repetition. Petri dish which has been poured the test solution then incubated for 24 hours in an incubator at 37°C. Then measured the inhibition zone formed around the agar wells by used a vernier caliper. Measurement data were analyzed with one way Anova test with a significance level of 0,05. Results: There were significant differences in the inhibition zone between pairs of test solution, except sarang semut extract 10% with sarang semut extract 20% and a positive control. Conclusion: Sarang semut extract has antibacterial activity against E. faecalis, extract concentration of 10% has the same antibacterial activity with extract concentration of 20% and 2,5% sodium hypochlorite. Extract with a concentration of 40% has the highest antibacterial activity, followed by a concentration of 20%, and the lowest is a positive control.