Introduction: In recent years, various methods such as lasers and different solutions have been investigated to eliminate microorganisms in the root canal. This investigation was aimed at comparing the bactericidal effect on Enterococcus faecalis between a diode laser with a wavelength of 940 nm and a mixture of a tetracycline isomer, an acid, and a detergent (MTAD). Methods: Forty single-rooted human lower premolar teeth were prepared for inoculation with E. faecalis and incubated for a period of 3 weeks. The 40 samples were randomly divided into 4 groups: the 940 nm diode laser group with irradiation through a 200 µm optical fiber with parameters of (1 W/CW,4 cycles, 20s interval); the MTAD group irrigated with 5 mL for 5 minutes; also, a positive group with 3% NaOCl and a negative group with 0.9% saline solution, both irrigated with 5 mL for 5 minutes. For statistical analysis, the Mann-Whitney U and Wilcoxon tests were used. Results: The 3% NaOCl positive control group had greater bacterial reduction after treatment (99.99%) compared to the 940nm diode laser (98.96%) and MTAD (99.34%) groups. Statistically, we found a significant difference between all groups regarding the reduction in the colony count (P<0.05). Conclusion: It was found that there was a significant difference in the percentage of reduction for the bacterial colonies between all the groups evaluated, with the greatest antibacterial effectiveness observed with 3% sodium hypochlorite.