OBJECTIVE The objective of this study was to compare in vitro the antibacterial activity of Tetraclean (mixture of doxycycline, citric acid and polypropylene glycol), Niclor 5 (5.25% sodium hypochlorite solution), Cloreximid (0.2% chlorhexidine and 0.2% cetrimide solution) and hydrogen peroxide 12 volumes on three endodontic pathogens associated with primary endodontic infections. Enterococcus faecalis, Streptococcus mutans and Staphylococcus aureus strains were selected to evaluate the antibacterial activity of endodontic irrigants by the agar disc-diffusion test. METHODS Paper discs were saturated with each one of the test solutions (at room temperature and preheating at 50°C) and placed onto culture agar-plates preadsorbed with bacterial cells and further incubated for 24 h at 37°C. The growth inhibition zones around each irrigant were recorded and compared for each bacterial strain. Kruskal-Wallis and Mann-Whitney tests were applied to compare the various groups. RESULTS At room temperature, Tetraclean showed significantly higher inhibition of bacteria growth than all other irrigants tested. Preheating at 50°C significantly increased growth inhibition for all the groups tested. At 50°C, hydrogen peroxide 12 volumes and Tetraclean showed significantly higher efficacy than all other irrigants tested. CONCLUSIONS 50°C-preheated hydrogen peroxide 12 volumes and Tetraclean showed highest inhibition of the bacterial growth.