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−2) and MB (100 µm) at different light doses. In vivo, mice were infected and 4 weeks later, randomly divided into three groups: control, APDT 1 (single session) and APDT 2 (two sessions of MB‐APDT). MB was used at 100 µm and energy dose was established at 150 J cm−2. 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−2. 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. Cutaneous leishmaniasis (CL) is a neglected disease that causes ulcerated lesions. Current treatments are limited due to high cost, resistance and toxicity. Photodynamic therapy has been put forward as an alternative treatment for CL. Here, we investigated methylene blue‐mediated photodynamic therapy (MB‐APDT) on Leishmania (L.) amazonensis in vitro using a red LED at different fluences. In vivo, we compared one and two MB‐APDT sessions at L. amazonensis‐induced CL in mice. Disease progress was evaluated by bioluminescence, lesion size and pain score. Our results indicate that MB‐APDT could be a cost‐effective strategy to apply in public health.