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Enterococcus faecalis is the most commonly found species in root-filled teeth evincing recalcitrant periradicular lesions and as a consequence, a role in causation of endodontic treatment failure has been suggested. The purpose of this study was to evaluate the prevalence of this bacterial species in root-filled teeth with or without periradicular lesions. Identification of E. faecalis was carried out by polymerase chain reaction (PCR) or conventional culture procedures. Overall, E. faecalis was detected by species-specific 16S rRNA gene-based PCR in 40/50 teeth (80%), while culture revealed the occurrence of this species in 8/50 teeth (16%). PCR was significantly more effective than culture in detecting this bacterial species (p < 0.001). Of 27 root-filled teeth with no periradicular lesions, E. faecalis was found in 22 cases (81.5%) by PCR and in five cases (18.5%) by culture. Of 23 root-filled teeth with periradicular lesions, E. faecalis was identified in 18 cases (78%) by PCR and in three cases (13%) by culture. Regardless of the identification technique used, no significant difference was observed when comparing the occurrence of E. faecalis in root-filled teeth with and without periradicular lesions (p > 0.05). Although these findings apparently put into question the status of E. faecalis as the main species causing endodontic treatment failure, other related factors still need to be clarified before this assumption turns into certainty.