The presence of Enterococcus faecalis in root canal teeth affected by primary and secondary periapical lesions was studied using polymerase chain reaction (PCR) assays. The association between presence of E. faecalis with clinical signs of apical lesions was assessed to evaluate a possible relationship between clinical findings. Microbial samples were obtained from healthy patients affected by different periapical lesions, 79 teeth with primary periapical lesion and 23 with secondary periapical lesion. For each tooth, clinical symptoms and X-ray appearance were examined. E. faecalis was detected in 6 of 79 samples with primary lesion (7.6%), and in 9 of 23 with secondary lesion (39.1%). Suggested association was found between E. faecalis and secondary apical lesions. As regard specific signs and symptoms E. faecalis was more associated with asymptomatic lesions (all p<0.05) than with symptomatic apical lesions. The study confirms the high presence of E. faecalis in secondary apical lesions. However, its effective role in endodontic pathogenesis such as bone periapical lesions needs to be clarified.