Objectives. The aim of this study was to determine whether irrigation with sodium hypochlorite, chlorhexidine, and ozone gas, alone or in combination, were effective against Enterococcus faecalis and Candida albicans; these are microorganisms frequently isolated from teeth with periapical lesions resistant to endodontic treatment. Material and Methods. 220 single root teeth, recently extracted, were inoculated with Candida albicans and Enterococcus faecalis. The formulations tested were sodium hypochlorite at 1, 3, and 5% chlorhexidine at 0.2% and 2% and ozone gas applied for different periods of time. The combination of sodium hypochlorite at 5% and chlorhexidine at 2%, with gaseous ozone, were also assessed. For the most active treatments the mechanism of action was assessed through flow cytometry. Results. Sodium hypochlorite, chlorhexidine, and gaseous ozone alone were ineffective in completely eliminating the microorganisms. The association of chlorhexidine at 2% followed by ozone gas for 24 seconds promoted the complete elimination of Candida albicans and Enterococcus faecalis. Flow cytometry shows that ozone and chlorhexidine act differently, which could explain its synergic activity. Conclusions. This new disinfection protocol, combining irrigation with chlorhexidine at 2% and ozone gas for 24 seconds, may be advantageous when treating infected root canals.