Objective: The aim was to determine the antimicrobial effect of ozonated water, ozonated water with ultrasonication, sodium hypochloride and chlorhexidine (CHX) in human primary root canals contaminated by Enterococcus faecalis (E. faecalis). Materials and Methods: Fifty-eight extracted human primary molar teeth were used. Crowns were cut off using a diamond saw under water-cooling. One hundred roots were obtained and mechanically prepared. The roots were then sterilized by autoclaving in water for 15min at 121°C. All samples were contaminated with E. faecalis for 24h and the root canals were randomly divided into five groups (n = 20). Group I: 25mg/L of Ozonated water (O3aq), Group II: 25mg/L of O3aq with ultrasonication, Group III: 2.5% Sodium hypochloride (NaOCl), Group IV: 2% CHX and Group V: Positive control. The canal of each specimen was irrigated for 4min and positive control was untreated. All root canals were agitated with sterile saline solution. The saline solution was collected from canals with sterile paper points. For each specimen, the paper points were transposed to eppendorf vials containing 2 ml of brain heart infusion. According to bacterial proliferation, the mean values of optical density were achieved by ELI˙SA (Biotek EL ×800, Absorbance Microplate Reader, ABD) and the data were analyzed. Results: NaOCI, CHX and two types of O3aq were found statistically different than positive control group. NaOCI irrigation was found significantly most effective. Conclusions: NaOCl, CHX and O3aq applications provide antibacterial effect in vitro conditions in primary root canals.