The objective of the present study was to investigate the presence of nine bacterial species in root-filled teeth associated with periapical lesions using a polymerase chain reaction analysis and to correlate these species with clinical features of the cases. DNA was extracted from 45 canal samples of root-filled teeth with periapical lesions. A PCR assay using species-specific primers of 16S rDNA and the downstream intergenic spacer region was used for microbial detection. Enterococcus faecalis was the most prevalent species, detected in 77.8% of the study teeth, followed by Peptostreptococcus micros, detected in 51.1%. Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Prevotella nigrescens were detected in 35.6%, 22.2%, 11.1%, and 11.1% of the sampled teeth, respectively. Moreover, PCR detected Filifactor alocis in 26.7%, Treponema denticola in 24.4%, and Tannerella forsythia in 4.4% of the samples. T. denticola and P. micros were statistically associated with tenderness to percussion (p < 0.05). P. nigrescens was associated with the presence of spontaneous pain and abscess (p < 0.05). P. endodontalis and P. nigrescens were associated with purulent exudates (p < 0.05). Synergistic relationship was also observed between some species. The results of this study indicated that E. faecalis was the most frequently identified test species by PCR in teeth with failing endodontic treatment.345537540