INTRODUCTION: Antimicrobial photodynamic therapy (APDT) is a well-known light-based technology that has been widely studied as an alternative approach to fight cutaneous leishmaniasis (CL). APDT induces lipid peroxidation in cellular membranes due to the generation of oxidative stress OBJECTIVES: In this study, we evaluated the role of 1,9- dimethylmethylene blue (DMMB)-mediated APDT on a wild-type (WT) and a miltefosine-resistant (MF) strain of Leishmania amazonesis and analyzed several cellular processes to get insights into the underlying mechanisms of APDT. MATERIALS AND METHODS: For this, APDT was carried out using red light (??= 670???12 nm) and promastigotes were exposed to different concentrations of DMMB at 8 J/cm2. Then, we measured mitochondrial potential and intracellular levels of reactive oxygen species (ROS) and analyzed quantitative lipidomics of the main phospholipid classes using electrospray-mass spectrometry. DISCUSSION AND RESULTS: As a result, we observed overproduction of ROS, mitochondrial membrane depolarization, and a rapid lipid remodeling immediately after APDT. Of note, MF showed a higher content in levels of phosphatidylcholine (PC) as compared to the WT line before treatment, which suggests it could be also involved in the MF resistance mechanism. In addition, results showed that after APDT, PC levels were substantially decreased, while new phospholipid species of phosphatidylethanolamine (PE) were increased. CONCLUSION: In conclusion, our data suggest DMMB-mediated APDT promoted a significant lipid peroxidation in the parasite's membrane of both strains, which failed to manage redox imbalance, thus resulting in cellular malfunction and death.