Introduction: Debridement of root canal using appropriately safe and effective irrigants is the key factor for long-term success. Purpose of this study was to compare the antibacterial effect of propolis with 5.25% sodium hypochlorite, and 2% chlorhexidine against enterococcus faecalis. Materials &Methods: In this study, 36 single-canal roots were used. The crown was removed and instrumentation was prepared by step-back technique, then teeth were sterilized and contaminated with E. Faecalis, and divided into four groups with 9 cases: group1: Propolis, group2: 5.25% sodium hypochlorite, group3: 2% chlorhexidine and group4: controls. Irrigants were injected by a 27-gauge syringe and roots were incubated in 37°C for one week. Sampling was done and inoculated to tryptone soy broth media, after 24 hours the turbidity was measured. Samples were also cultured on agar plates, and colony-forming units were counted as CFU/ml. Data were analysed using the Mann-Whitney test. Results: The difference between propolis with mean value of 246.77 colonies and chlorhexidine with mean value of zero colonies, was significant (P=.002). Similarly, the difference between chlorhexidine and sodium hypochlorite with mean value 203.55 of colonies was significant and they had significant difference in turbidity (P=.002), too. No significant difference was observed between propolis and sodium hypochlorite with regard to the induced colonies (P=0.781) and their turbidity (P=0.495). Conclusion: It can be concluded that antibacterial activity of 2% chlorhexidine against E. faecalis is more obvious than propolis or 5.25% sodium hypochlorite. But antibacterial activity of propolis over 5.25% sodium hypochlorite or vice versa was not confirmed.