Cutaneous leishmaniasis (CL) is a neglected disease that promotes destructive lesions. Difficulties in treatment are related to accessibility of drugs, resistance and toxicity. Antimicrobial photodynamic therapy (APDT) has been emerging as a promising treatment for CL. In this work, we evaluated methylene blue (MB)-mediated APDT (MB-APDT) on Leishmania amazonensis in vitro and in vivo by bioluminescence technique. In vitro, MB-APDT was performed using a red LED (λ= 660±11 nm, 100 mW/cm ) and MB (100 µM) at different light doses. In vivo, mice were infected and 4 weeks later, randomly divided into 3 groups: control, APDT 1 (single session) and APDT 2 (2 sessions of MB-APDT). MB was used at 100 µM and energy dose was established at 150 J/cm . Parasite burden, lesion size and pain were evaluated weekly for 4 weeks. In vitro, lethal dose for 90% parasite inactivation was achieved at 48.8 J/cm . In vivo, although APDT 1 and APDT 2 groups have showed similar parasite burden after 4 weeks, two sessions were clinically better, especially considering the inflammatory process associated to CL. Our findings reinforce MB-APDT as a cost-effective treatment to combat CL.