This study determined the presence of microorganisms by culture and polymerase chain reaction in asymptomatic root-filled teeth with periradicular lesions. Furthermore, a disinfecting regimen using sodium hypochlorite (NaOCl), ethylenediaminetetraacetic acid (EDTA), chlorhexidine digluconate (CHX) irrigation, and calcium hydroxide (Ca(OH)(2)) dressing was assessed. After removal of the root-filling material, specimens of 20 cases undergoing retreatment were sampled. Moreover, the canals were sampled after each step of the disinfecting regimen. Prevalence of microorganisms was 60% by culture and 65% by polymerase chain reaction. In four of those samples (31%), DNA of Enterococcus faecalis was found. After further root canal preparation and irrigation using NaOCl and EDTA, microorganisms could be detected in none of the teeth. Thus, CHX and Ca(OH)(2) could not show further disinfection. In contrast, microorganisms were found in two teeth after the interappointment dressing. It may be concluded that proper root canal preparation and irrigation using NaOCl and EDTA are sufficient for decontamination of the root canal system during endodontic retreatment.