The purpose of this study was to evaluate, in vitro, the antimicrobial effect of iRoot BP, iRoot BP Plus, and mineral trioxide aggregate (MTA) against Entercoccus faecalis and Candida albicans by using direct contact test. The materials were tested immediately after application to the microtiter wells and after setting for 1-day and for 7-days. Ten microliters of microbial suspension was added to each well for direct contact with each material for 1 h at 37°C and 100% humidity. Then fresh media was added and, survival of bacteria and fungi was determined by using 10-fold serial dilution and inoculated onto agar plates. In fresh and 1-day samples all of tested materials showed statistically significant antimicrobial effects compared to control groups (p<0.05). In 7-day samples, there were no significantly differences compared to control groups. MTA, iRoot BP and iRoot BP Plus had similar antimicrobial efficacy against E. faecalis and C. albicans.