Photodynamic inactivation (PDI) has been attracting attention as an innovative technology to treat topical diseases, such as cutaneous leishmaniasis (CL) and infections caused by multidrug-resistant microorganisms. Zn(II) mesotetrakis( N-n-hexylpyridinium-2-yl)porphyrin (ZnTnHex-2-PyP4+) is a lipophilic water-soluble Zn(II) porphyrin with improved photophysical properties, high chemical stability, and cationic/amphiphilic character that can enhance its interaction with cells. Thus, this study aimed to investigate the PDI effects mediated by ZnTnHex-2-PyP4+ on Leishmania amazonensis. Confocal fluorescence microscopy was explored to study the interaction of ZnTnHex-2- PyP4+ with promastigotes. The PDI action was analyzed by cell membrane integrity, mitochondrial membrane potential (????m), and cell morphology. Promastigotes were incubated with ZnTnHex-2-PyP4+ for 5 min at 0.62 and 1.25 ??M and irradiated by a LED (410 nm) for 1 or 3 min (2.3 and 3.4 J/cm2, respectively). PDI on amastigotes and the cytotoxicity on macrophages were also analyzed (3.4 J/cm2). Fluorescence microscopy revealed that parasites efficiently uptake ZnTnHex-2-PyP4+ and displayed a punctate labeling pattern along with the cytoplasm. An intense ????m depolarization was also observed, which in association with microscopy results, suggests that ZnTnHex-2- PyP4+ may accumulate in the mitochondrion, or other well-defined structures close to it. Moreover, ZnTnHex-2-PyP4+ at concentration as low as 0.62 ??M led to the immediate inactivation of >95% of promastigotes, regardless of the light dose used. Loss of the fusiform shape and plasma membrane wrinkling were also observed. After a single treatment session in amastigotes, PDI led to a reduction of 70% in the infection index. No considerable toxicity was observed on mammalian cells. Thus, PDI of Leishmania parasites showed in vitro efficiency at a submicromolar concentration of ZnTnHex-2-PyP4+, with short pre-incubation and irradiation times. The results encourage further studies in CL pre-clinical assays and PDI of other microorganisms.