Objective: The purpose of this study was to investigate the antibacterial effects of two different types of laser and aqueous ozone in human root canals infected by Enterococcus faecalis. Background data: Many techniques have been developed to find an alternative to sodium hypochlorite as a disinfection agent for infected root canals. However, no study has evaluated the exactly the same antimicrobial agent with sodium hypochlorite (NaOCl). Methods: Eighty mandibular premolar teeth with single roots and canals were selected. Following root canal preparation and irrigation, sterilization was performed in an autoclave. E. faecalis was incubated in the root canals and kept at 37 degrees C for 24 h. The teeth contaminated with E. faecalis were divided into one negative control group (NaOCl) and three experimental groups; (Er: YAG laser, KTP laser, and aqueous ozone groups)(n = 20). A disinfection procedure was performed for 3 min in order to standardize all groups. After this procedure, the microbial colonies were counted. Results: The results indicated that whereas the NaOCl group exhibited the highest antibacterial effect among all groups, the aqueous ozone showed the highest antibacterial effect among the experimental groups. Whereas a statistically significant difference was noted between the aqueous ozone and laser groups (p < 0.05), the difference between the Er: YAG and KTP lasers was not statistically significant (p > 0.05). Conclusions: The results showed that when aqueous ozone was applied with the aim of disinfecting the root canals, it exhibited a higher antibacterial effect than the KTP and Er: YAG lasers. However, the antibacterial effect of the aqueous ozone was insufficient when compared with NaOCl.