In this study, microbial diversity of the root canal microbiota related to different endodontic infections was investigated. In total, 45 patients with endo–perio lesions (8 patients), chronic periapical periodontitis (29 patients) and pulp necrosis (8 patients) were recruited. In 19 (42.2%) patients there was secondary infection of root canals. Microbial specimens were collected from root canals of non-vital teeth with or without changes in periapical area visible in X-ray. Then, oral microbiota were detected and identified using the culture method and real-time PCR amplification primers and hydrolysis-probe detection with the 16S rRNA gene as the target. Overall, 1434 species/genes from 41 different genera of 90 various microbial species were retrieved. Of the major reported phyla, Firmicutes (62.9%), Actinobacteria (14.0%), Bacteroidetes (12.1%), Proteobacteria (9.1%) and Fusobacteria (4.2%) were detected. Of the bacterial species, 54.6% were strict anaerobes. Corynebacterium matruchotii (p = 0.039) was present significantly more frequently in chronic periapical periodontitis. Moreover, the higher values of Decayed, Missing and Filled Permanent Teeth index were positively correlated with relative abundance of Actinomyces spp. (p = 0.042), Lactobacillus spp. (p = 0.006), Propionibacterium spp. (p = 0.024) and Rothia spp. (p = 0.002). The multivariate analyses revealed differences in total root canal samples, where components that affected grouping of root samples into four main categories were identified. Anaerobic Gram-negative bacteria predominated in root canals of teeth with pulp necrosis and periapical lesions. Facultative anaerobic Gram-positive bacteria predominated in canals with secondary infections. All detected members of mixed population groups that might serve as keystone species contributed to the entire community in its clinical relevance.