Background: An improved light activated disinfection technique utilizing a specific photosensitizer formulation, liquid optical-conduit, oxygen-carrier and light energy of appropriate wavelength has been introduced recently. This study tested the efficacy of this improved light activated disinfection on ex vivo biofilms of Enterococcus faecalis at two different stages of maturation. Methods: Eighty-five tooth sections were prepared and endodontic biofilm of E. faecalis were grown within the root canal. In stage 1, conventional light activated disinfection (LAD), chemical disinfectant (sodium hypochlorite) and improved LAD were tested on four-day-old (immature) biofilms. In stage 2, conventional LAD, improved LAD and chemomechanical disinfection (alone and in combination with improved LAD) were tested on four-week-old (mature) biofilms. Results: Sodium hypochlorite and improved LAD showed the ability to significantly inactivate bacteria in four-day-old biofilms when compared to the control and LAD (p < 0.05). Inactivation of bacteria from deeper dentine was higher in improved LAD than sodium hypochlorite. In four-week-old biofilms, a combination of chemomechanical disinfection and improved LAD produced significant bacterial killing compared to either chemomechanical disinfection or improved LAD alone. Conclusions: This study highlighted the potential of improved LAD to kill bacteria within dentinal tubules. In combination with chemomechanical preparation, the improved LAD significantly inactivated four-week-old biofilm bacteria.