Limitations in endodontic disinfection are collectively due to the biofilm mode of bacteria in root canal systems, anatomical complexities, dentin structure/composition, and limitations associated with chemical disinfectants. Consequently, advanced disinfection strategies are developed and tested in Endodontics. The primary aim of these advanced anti-biofilm strategies is to eliminate biofilm bacteria from the uninstrumented portions and anatomical complexities of the root canal system without inducing untoward effects on healthy tissues. Here various challenging factors in root canal disinfection and different advanced therapeutic strategies for endodontic biofilms such as antibacterial nanoparticles, antimicrobial photodynamic therapy, laser-assisted root canal disinfection, ozone, and herbal/enzyme alternatives have been discussed.