The product of the surface-related development of microorganisms is the most common endodontic infection. The biofilm of the root canal is a very complicated hierarchical culture, and in experiments, it is hard to replicate its attributes. The difficulty is due to the complicated anatomy and the existence of the biofilm in the root canal systems. This effect may be simplified by research on mono-species or dual-species biofilms and may not be a direct representation of clinical reports' results