BACKGROUND Periapical lesions are primarily caused by infections in the root canals. The objective of this study was to assess the antibacterial effectiveness of diode laser during root canal treatment in artificial models of infected periapical lesions. MATERIAL AND METHODS One hundred twenty-two extracted premolar single-rooted teeth were inserted into methyl methacrylate artificial models of periapical lesions, and bacterial solutions of Enterococcus faecalis (ATCC 29212) and Streptococcus mitis (ATCC 49456) were then applied to the models. The respective diameters of lesions in the artificial models represented 3 different subgroups based on lesion size. The laser protocol used for endodontic disinfection had a power output of 1.5 W and a wavelength of 810 nm. The impact on cell viability was evaluated by flow cytometry. RESULTS Disinfection with laser did not differ between microorganisms (P=0.137), and laser irradiation with a longer duration had better disinfecting action for both microorganisms (P.