Aim: To evaluate the antibacterial effect of diode laser, associated or not with 2.5% sodium hypochlorite (NaOCl), against Enterococcus faecalis. Materials and methods: Eighty dentin blocks were obtained from single-rooted human teeth and sterilized. Seventy were inoculated with 0.01 mL of fresh bacterial inoculum (within 24 hours of preparation from pure culture) standardized to 1 McFarland turbidity. Contaminated blocks were incubated for 7 days at 37°C in humid conditions. Ten uncontaminated samples were incubated at 37°C during the contamination period to serve as a negative control group, while 10 of the infected specimens served as a positive control group. The dentin blocks were randomly divided into eight experimental groups (n = 10 each) according to the method of decontamination: 2.5% NaOCl alone; 2.5% NaOCl + photodynamic therapy (PDT) with methylene blue/660 nm laser at 18 J for 180 seconds; 2.5% NaOCl + PDT with methylene blue/660 nm laser at 8 J for 80 seconds; methylene blue alone; PDT alone with methylene blue/660 nm laser at 18 J for 180 seconds; PDT alone with methylene blue/660 nm laser at 8 J laser for 80 seconds; positive control group; and negative control group. Microbial growth was evaluated by culture medium turbidity and microbial concentration was analyzed by UV spectrophotometry (adjusted to read at wavelength l = 600 nM). Results: Root canals treated with laser alone at 18 J for 180 seconds had higher bacterial contamination compared with groups in which NaOCl was used, with or without laser irradiation at 18 J for 180 seconds (p < 0.05). Conclusion: Photodynamic therapy with a 660 nm diode laser effectively reduced E. faecalis contamination. These findings can guide development of further studies in search of better alternatives for endodontic treatment. Clinical relevance: Chemical and mechanical root canal preparation plays an essential role in reducing microbial burden. However, microorganisms present in areas not mechanically reachable by endodontic instruments. As an alternative to fix this problem, the laser can be applied.