Amongst the bacterial species which most often cause endodontic failures, Enterococcus faecalis is the most important. This study compared the effectiveness of sodium hypochlorite and two new generation antibiotic-based endodontic irrigants, Tetraclean and MTAD. By means of an in vitro agar dilution assay, we show that both Tetraclean and MTAD are 100% effective against 54 clinical isolates at dilutions up to 1:256 and 1:1048, respectively, whereas sodium hypochlorite completely loses its effectiveness when diluted more than 32 times. The bactericidal effect of both Tetraclean and MTAD can be ascribed not just to their antibiotic component per se, but also to a synergistic effect among the several ingredients included in the formulations. Moreover, by an ex vivo model of teeth extracted and experimentally infected with E. faecalis ATCC 29212, we show that both the antibiotic-based endodontic irrigants are effective in eliminating bacterial cells in 93 to 100% of the test samples. The results of these pre-clinical studies strongly support a wider use of this new group of endodontic irrigants in daily clinical practice.