Enterococcus faecalis is frequently recovered from refractory endodontic infections and has been implicated in endodontic treatment failures. This study compared real-time quantitative PCR (qPCR) assay to cultivation for E. faecalis detection and quantitation during endodontic treatment. A reverse-transcription PCR (RT-PCR) assay was also developed to detect the bacterium clinically in the viable but nonculturable (VBNC) state. Intra-canal samples (n = 87) were collected upon access, post-instrumentation/irrigation and postcalcium hydroxide treatment from 15 primary and 14 refractory infections involving 29 single-rooted teeth, and analyzed by the three methods. The bacterium was up to three times more prevalent in refractory than primary infections at each collection step. Overall, qPCR detected significantly more E. faecalis-positive teeth and samples than cultivation (p < 0.001). VBNC E. faecalis was detected by RT-PCR in four samples that were negative by cultivation. These findings support qPCR and RT-PCR as more sensitive methods than cultivation for detecting E. faecalis in endodontic infections.