INTRODUCTION: The aim of this study was to compare the antimicrobial effects of 2.5% Sodium hypochlorite (NaOCl), 2% Chlorhexidine Gluconate (CHX) and BioPure MTAD (MTAD) on Enterococcus (E) faecalis-contaminated root canals of human extracted teeth. MATERIALS AND METHODS: Seventy human intact extracted single-rooted teeth with straight root canal randomly divided into 5 groups: positive control (n=5), negative control (n=5), 2.5% NaOCl (n=20), 2% CHX (n=20), and MTAD (n=20). Each tooth was instrumented using the passive step-back technique hand and rotary instruments. E. faecalis incubated into the canals and grew for 4 weeks. Canals irrigated using three mentioned solutions for 5 minutes. Samples were taken from canal walls and transferred into Brain Heart Infusion (BHI) culture medium and placed in an incubator at 37ºC for 96 hours and bacteriological evaluations were done. Chi- Square test and SPSS software were used for the statistical analysis of the results. RESULTS: Bacterial growth was seen in only one sample of MTAD group (5%), but in 4 of CHX group (20%) and 5 of NaOCl group (25%). Chi-Square test showed no statistically difference between groups. CONCLUSION: Based on the results of this study, it seems that all three solutions have acceptable antimicrobial effect on E. faecalis.