Biofilms located within the root canals of teeth are a unique and pressing concern in dentistry and in medical microbiology. These multispecies biofilms, which include fungi as well as bacteria, form in a protected site with low shear stress and low oxygen tension. Systemic antibiotics are of limited value because of the lack of blood flow of the site, and issues with innate and acquired resistance. Physical disruption using hand or rotary powered instruments does not reach all locations in the root canal system where biofilms are present. Alternative strategies including agitated irrigation fluids, continuous chelation, materials with highly alkaline pH, and antimicrobial nanoparticles are being explored to meet the challenge. Detection and quantification of biofilms using fluorescence-based optical methods could provide an indication of successful biofilm removal and an endpoint for physical and chemical treatments.