Biofilm refers to the complex, sessile communities of microbes found either attached to a surface often buried firmly in a self-produced extracellular matrix. Cells within a biofilm have intrinsic characteristics that are different from those of planktonic cells, making them tolerant to severe conditions and resistant to antibacterial treatments. Bacterial interactions also play a key role in biofilm formation and community behavior. Therefore, eradication of biofilms is very challenging. Although fungi, Archaea and viruses have been detected in teeth with pulp necrosis and apical periodontitis, bacteria are the main components of endodontic infections. Bacterial abundance and diversity in the root canals may vary according to different types of endodontic diseases and to different clinical conditions (e.g., symptomatology); and mainly determined by ecological factors and still unknown microbial interactions. Many approaches have been made to improve the disinfection levels of the endodontic treatment and promote healing of apical periodontitis. However more preclinical studies and well-designed multicenter clinical trials are needed to evaluate the prospects of these strategies. Here, we address the composition of intraradicular biofilms and their response to endodontic disinfection strategies. In addition, extraradicular biofilms, which are rarer conditions, are also discussed.