Microbial biofilms are clusters of microorganisms immobilised on a surface or an interface, enveloped in a slimy matrix of their own creation. In infected root canals, microbial biofilms are formed on hard tissue surfaces of the root canal system. Infection of the root canal system is the cause of apical periodontitis, and complete elimination of the infection is the ultimate goal of root canal treatment. However, this aim is quite difficult, if not impossible, to achieve, due to not only the complexity of the root canal system, which offers inaccessible niches for microorganisms, but also the fact that there are protective advantages in residing in biofilms. Microorganisms in biofilms are more resistant to antimicrobial regimes than their counterparts in planktonic form. A good understanding of the distinctive features of a microbial biofilm, particularly in root canal infection, is important to enable development of techniques to eliminate the microbial biofilm, and as a result, (post-treatment) apical periodontitis. In the following review, the definition, main features and significance of a microbial biofilm, and its relevance to root canal infection will be discussed. Keywords: biofilm, endodontic, root canal infection