Introduction: The aim of this in vitro study was to compare the effectiveness of two intracanal medicaments and diode laser in disinfecting the root canal of experimentally infected human teeth with Enterococcus faecalis (EF). Materials and Methods: eighty extracted human single-rooted teeth were used. After root canal preparation and sterilization, the teeth were infected with EF. Then root canals were filled with one of three different disinfectants: 2% Chlorhexidine (CHX), calcium hydroxide paste Ca(OH)2 , a mixture of Ca(OH)2 and CHX or treated with diode laser (n=20 in each group). Antimicrobial assessments were performed at 2 and 7 days (n=10 in each time period). Microbial samples were obtained from root canals before and after the experiment. The data was analyzed using one- Way ANOVA test. Results: laser treatment showed statistically significant difference when compared Ca(OH)2 or CHX either for 2 days or 7 days (P-value≤ 0.05). diode laser showed higher reduction percentage . Conclusion: According to the results of this in vitro study, diode laser ,2% CHX and calcium hydroxide paste are all effective disinfectants.