Abstract Introduction Chronic apical periodontitis (CAP) is a frequent condition that has a considerable effect on a patient's quality of life. We aimed to reveal root canal microbial communities in antibiotic-naive patients by applying Illumina sequencing (Illumina Inc, San Diego, CA). Methods Samples were collected under strict aseptic conditions from 12 teeth (5 with primary CAP, 3 with secondary CAP, and 4 with a periapical abscess PA) and characterized by profiling the microbial community on the basis of the V6 hypervariable region of the 16S ribosomal RNA gene by using Illumina HiSeq2000 sequencing combinatorial sequence-tagged polymerase chain reaction products. Results Root canal specimens displayed highly polymicrobial communities in all 3 patient groups. One sample contained 5–8 (mean = 6.5) phyla of bacteria. The most numerous were Firmicutes and Bacteroidetes , but Actinobacteria , Fusobacteria , Proteobacteria , Spirochaetes , Tenericutes , and Synergistetes were also present in most of the patients. One sample contained 30–70 different operational taxonomic units; the mean (± standard deviation) was lower in the primary CAP group (36 ± 4) than in the PA (45 ± 4) and secondary CAP (43 ± 13) groups ( P  < .05). The communities were individually different, but anaerobic bacteria predominated as the rule. Enterococcus faecalis was found only in patients with secondary CAP. One PA sample displayed a significantly high proportion (47%) of Proteobacteria , mainly at the expense of Janthinobacterium lividum. Conclusions This study provided an in-depth characterization of the microbiota of periapical tissues, revealing highly polymicrobial communities and minor differences between the study groups. A full understanding of the etiology of periodontal disease will only be possible through further in-depth systems-level analyses of the host-microbiome interaction.