Enterococcus faecalis has long been mentioned in increased frequency with regard to teeth with post-treatment disease (PTD). The prominence of E. faecalis in root-filled teeth with apical periodontitis has made it a focus of attention as an etiological factor of PTD. To evaluate and compare bacterial culture and species-specific polymerase chain reaction for the detection of enterococcus faecalis in retreatment cases with apical periodontitis in rural Indian population. A total of 80 samples were obtained from 40 patients, divided into 2 groups and analyzed by the two methods. The samples were grown under anaerobic conditions during culture and the determination of the microbial species was based on biochemical tests. DNA was extracted and analyzed for PCR using species-specific primer. The association of microbiological findings with clinical features was investigated. Enterococcus faecalis was isolated from 46.5% of the selected teeth. Culture and PCR detected the test species in 25% and 67.5% of the samples. PCR was significantly more effective than culture in detecting this bacterial species. E.faecalis was clearly proven as the causative factor of apical periodontitis. Therefore, combining molecular and culture technique is probably the best approach available to provide comprehensive and a panoptic information about the microflora associated with endodontic infections.