This present study attempted to identify the antibacterial effects of aqueous and gaseous ozone in human root canals that are contaminated with Pseudomonas aeruginosa. The differences and antibacterial effects of gaseous and aqueous ozone (manual and ultrasonic) have not been compared to each other yet in any studies. Eighty single-root premolar teeth were prepared and then disinfected and sterilized. P. aeruginosa were incubated in root canals and kept at 37°C for 24 h. The root canals were contaminated with P. aeruginosa divided into 1 positive control, sodium hypochlorite (NaOCl) and three experimental groups: aqueous ozone with manual technique, aqueous ozone with ultrasonic technique and gaseous ozone (n=10). Disinfection procedures were performed for 5 min in order to ensure standardization among all working groups. Remaining microorganism colonies were counted on blood agar plates. Then, data were evaluated and statistically analyzed by one-way ANOVA and Tukey's test. As a result, although there were no statistically significant differences between the three groups (NaOCl, aqueous ozone with manual, and ultrasonic technique) (P>0.05), there were statistically significant differences between the gaseous ozone and all other groups (P<0.05). Both the manual and ultrasonic techniques of aqueous ozone achieved complete elimination of P. aeruginosa in root canals.