Antimicrobial photodynamic therapy (APDT) combined with endodontic treatment has been recognized as an alternative approach to complement conventional root canal disinfection methods on bacterial biofilms. We developed an in vitro model of bioluminescent Candida albicans biofilm inside curved dental root canals and investigated the microbial reduction produced when different light delivery methods are employed. Each light delivery method was evaluated in respect to the light distribution provided inside curved root canals. After conventional endodontic preparation, teeth were sterilized before canals were contaminated by a bioluminescent strain of C. albicans (CEC789). Methylene blue (90 μM) was introduced into the canals and then irradiated (λ = 660 nm, P = 100 mW, beam diameter = 2 mm) with laser tip either in contact with pulp chamber or within the canal using an optical diffuser fiber. Light distribution was evaluated by CCD camera, and microbial reduction was monitored through bioluminescence imaging. Our findings demonstrated that the bioluminescent C. albicans biofilm model had good reproducibility and uniformity. Light distribution in dental tissue was markedly dependent on the light delivery system, and this strategy was directly related to microbial destruction. Both light delivery systems performed significant fungal inactivation. However, when irradiation was performed with optical diffuser fiber, microbial burden reduction was nearly 100 times more effective. Bioluminescence is an interesting real-time analysis to endodontic C. albicans biofilm inactivation. APDT showed to be an effective way to inactivate C. albicans biofilms. Diffuser fibers provided optimized light distribution inside curved root canals and significantly increased APDT efficiency.