Background and aim: Colonization of residual organisms in the root canal are major causes of root canal treatment failure. Therefore, the effective removal of organisms during root canal cleaning stages is of great importance. In this study, we aimed to investigate and compare the antimicrobial effects of several methods including high power laser irradiation, photodynamic therapy and triple antibiotic paste (TAP) on Enterococcus faecalis in the root canal. Materials and methods: For the present laboratory study, 80 anterior single canal teeth were randomly divided into seven experimental groups (970 nm diode laser irradiation, 445 nm diode laser irradiation, 660 nm photodynamic therapy with doxycycline as a photosensitizer, TAP, 970 nm diode laser combined with TAP, 445 nm diode laser combined with TAP and sodium hypochlorite groups), each containing 10 teeth, and two positive and negative control groups, each containing five teeth. Afterwards, an E. faecalis suspension was prepared and injected into the root canals of all groups (except the negative control group), and after using the desired lasers or drugs and incubating for the necessary time, the grown colonies were counted and significance level of less than 0.05 was considered. Results:Among seven experimental groups, in those that included triple antibiotic paste (TAP, 970 nm diode laser with TAP, and 445 nm diode laser with TAP groups), no bacteria grew, while the 970 nm and 445 nm diode laser groups had the highest bacterial growth. Statistically, all studied groups were effective, and the results showed a statistically significant difference between groups (P value < 0.05). Conclusion: The findings of the present study show that all of the above-mentioned methods were effective enough for bacterial reduction. Triple antibiotic paste was proved to achieve complete bacterial elimination. Photodynamic therapy with doxycycline as a photosensitizer was shown to provide significant results of bacterial reduction and diode laser irradiation at both wavelengths (970 nm and 445 nm) were also reported to have antibacterial effect, although slightly lower than the previous methods.