Objective: To compare the reduction of E. faecalis counts in root canals produced by irrigation with distilled water, hydrogen peroxide, sodium hypochlorite, chlorhexidine, and combinations of solutions, in vitro. Study Design: Study sample included sixty mandibular premolar teeth mounted in dental stone. Root canals were prepared using crown down technique under distilled water irrigation. Specimens were sterilized overnight by ethylene oxide gas. Each canal was completely filled up with the E. faecalis suspension and incubated at 37ºC for 24 hours. Root canals were irrigated with the test solutions. A volume of 5 ml of the tested irrigant was used for each sample with an exposure period of 5 minutes. 10 ml of distilled water was used as final irrigant in all the samples. Canals were sampled before and after irrigation. After serial dilutions, samples were plated onto Mitis salivarius agar, and the colony forming units were counted. Results were analyzed by Chi-square test, Fisher's exact test, and one-way ANOVA followed by multiple range Tukey HSD test with the level of significance set at P < 0.05. Results and Conclusions: Reduction of colony counts in distilled water group was significantly lower than the mean reduction in all the other groups. However, no other contrasts are statistically significant. Combination of sodium hypochlorite and chlorhexidine showed the most effective antimicrobial activity followed by sodium hypochlorite and hydrogen peroxide together. Hydrogen peroxide was the least effective irrigant when used alone.