Aim: To isolate and identify in a Chilean population, Enterococcus spp. from root canals with persistent chronic apical periodontitis (CAP) and to investigate the potential correlation between the bacteria and the observed clinical features. Methods: Twenty patients with indication for endodontic retreatment due to persistent CAP were selected. Data from patient general health and dental clinical history were recorded. During retreatment, a microbial sample was obtained from the root canal and inoculated in a selective Enterococcus medium. Using bacterial cultivation methods, bacterial isolates belonging to the genus Enterococcus were identified. The relationship between the number of colony-forming units of Enterococcus spp. and patient clinical data was assessed statistically by the Pearson Chi square and Fisher exact tests. Finally, a Polymerase Chain Reaction (PCR) assay to determine the most prevalent species of Enterococcus spp. was conducted in the clinical samples, and the results were analyzed by a proportion comparison test. Results: Enterococcus spp. strains were isolated in 70% of the patients. Most of them (98.8%) accounted for Enterococcus faecalis and only 1.2% for Enterococcus faecium. A high frequency of E. faecalis was found in teeth with inadequate endodontic treatment or dental crown restorations. Conclusions: This study concluded that E. faecalis is prevalent in root canals with persistent CAP in a Chilean population. E. faecium as found in a single case with the poorest root canal filling. Further studies are still required to investigate the presence of other species, which may be linked to persistent chronic apical periodontitis.