Apical periodontitis and its different manifestations are caused primarily by bacterial infection of the root canal system. Bacteria are usually organized in biofilm communities and can colonize not only the main canal but also spread to other areas in the root canal system. In these regions, bacteria are more difficult to reach and eliminate during treatment. The knowledge of endodontic infections has substantially increased over the past 4 decades, including the main species involved in the different forms of disease, their virulence factors, interactions, ecology, organization and spatial distribution in the root canal system, patterns of antimicrobial resistance, and so on. However, there is a need for all of this information to be translated into improvements in clinical practice and treatment outcomes. This article reviews the present status of endodontic microbiology, discusses perspectives for future research and directions, and emphasizes the need for a call to action in the field of applied endodontic microbiology.