INTRODUCTION: The aim of the present study was to investigate the composition of the root canal microbiota in endodontic failures in order to identify and quantify these microorganisms. METHODS: Microbiological samples were taken from 36 root canals with persistent endodontic infection. The presence, levels, and proportions of 79 bacterial species were determined by checkerboard DNA-DNA hybridization. The Pearson correlation coefficient was used to investigate the relations between bacterial counts and clinical conditions (P ≤ .05). RESULTS: Enterococcus faecium (36%), Streptococcus epidermidis (36%), Eubacterium saburreum (28%), Parvimonas micra (28%), Streptococcus sanguis (28%), Capnocytophaga sputigena (28%), Leptotrichia buccalis (28%), Enterococcus faecalis (28%), and Staphylococcus warneri (28%) were the most prevalent species; and there was a low prevalence of Treponema socranskii (3%), Fusobacterium periodonticum (3%), Capnocytophaga gingivalis (3%), and Spiroplasma ixodetis (3%). The highest mean levels were found for the following species: E. faecium, Dialister pneumosintes, Staphylococcus epidermidis and Helicobacter pylori. There was a statistically significant difference between the levels of gram-negative species and gram-positive species (13.5 × 10(5) vs 6.5 × 10(5), respectively). A positive correlation was found between the area of the periapical lesion and the levels of gram-negative and rod species (P < .05). CONCLUSIONS: The microbiota from teeth with persistent apical periodontitis presents a mixed and complex profile, hosting E. faecium and S. epidermidis as the most highly prevalent species. No correlation was found between any of the species tested and clinical findings; however, periapical lesions with the largest areas presented higher counts of gram-negative and rod species.