The objective was to compare the antibacterial effects of adjunctive disinfection using diode laser and gaseous ozone compared to the medical dressings calcium hydroxide (Ca(OH) 2) and chlorhexidine gel (CHX-Gel) on Enterococcus faecalis biofilms in human root canals ex vivo. Root canals of 180 human extracted teeth were infected by E. faecalis and divided into 3 main groups (G): G1, control; G2, instrumentation and irrigation using 0.9% NaCl; G3, instrumentation and irrigation using 1% NaOCl. In each main group, the following treatments were applied: gaseous ozone, diode laser, and medical dressings of Ca(OH) 2 or CHX-Gel for 7 days ( n = 15). Reduction of colony forming units (CFUs) inside the root canal of planktons and frequencies of adherent bacteria after treatment were calculated. Bacterial reduction was significantly affected by the irrigation protocol ( p < 0.0005) and the disinfection method ( p < 0.0005), and a significant interaction between both factors could be observed ( p < 0.0005; ANOVA). In G3 (instrumentation using 1% NaOCl), no significant effect of disinfection methods could be demonstrated on planktonic bacteria ( p = 0.062; ANOVA) and frequencies of adherent bacteria ( p > 0.05; chi-square test). Instrumentation and irrigation using NaOCl combined with ozone or laser application resulted in comparable bacterial reduction on E. faecalis to the application of medical dressings.