Inflammatory reaction in the periapical tissues is induced by microbial infection in the root canal system. The aim of root canal treatment is to preserve healthy periapical tissues or to provide healing of them in restorable teeth, which have sufficient periodontal support. The amount of microbial cells in the root canal system and their virulence as well as host responses influence on the degree of periapical inflammation and symptoms. Microbial biofilm formation is typically seen on root canal walls but some microbial species are able to invade the dentine tubules to varying depth. In prolonged and complicated infections, or in case of risk of systemic spread of infection, root canal sampling for microbiological diagnostics is recommended. Anaerobic gram-negative rods are commonly isolated organisms in primary infections. In post-treatment disease, the microflora is dominated by facultatively anaerobic gram-positive cocci and rods such as Streptococcus, Enterococcus, Peptostreptococcus and Actinomyces species. Instrumentation, disinfection and interappointment medication in strict aseptic conditions are essential steps for eradication of microbial species from the infected root canal system. During past decades, Enterococcus faecalis and Candida albicans have been commonly associated organisms in treatment-resistant infections. Novel microbial detection methods are giving increasing knowledge about microbial species associated with endodontic infections and their roles in them.