Aim:To comparatively assess the antibacterial effectiveness of 5% sodium hypochlorite, 940 nm diode laser, and gaseous ozone against Enterococcus faecalis biofilm produced on the substrate. Materials and methods:Freshly extracted maxillary central incisor teeth of human source were decoronated and vertically sectioned after biomechanical preparation.The samples were then, after sterilization, placed in Eppendorf tubes filled with 1 mL of bacterial solution containing 1.5 × 10 5 colony-forming units (CFUs)/mL of E. faecalis. Then, these adulterated samples were divided into four groups (n = 8) depending upon the method of disinfection used: group I, 5% sodium hypochlorite (positive control); group II, normal saline (negative control); group III, gaseous ozone; and group IV, 940 nm diode laser.At the conclusion of 3 weeks, all the samples were disinfected according to their groups and were analyzed qualitatively and quantitatively. Results:The positive control group (5% sodium hypochlorite) showed statistically significant results in comparison with the other three groups (p < 0.05). Statistically, there was no significant difference found between the experimental groups, i.e., ozone group and diode laser group (p > 0.05). Conclusion:A 5% sodium hypochlorite showed the highest antibacterial effect against E. faecalis biofilm formed on substrate, i.e., tooth. Both diode laser and gaseous ozone groups have a statistically significant antibacterial action on the infected root canals.Efficacy of 5% Sodium Hypochlorite, 940 nm Diode Laser, and Gaseous Ozone against Enterococcus faecalis Biofilm formed on Tooth Substrate: A Scanning Electron Microscope Study.