Cutaneous leishmaniasis is the most common form of leishmaniasis and the available chemotherapy causes serious side effects, justifying the search for new therapies. This study investigated the antileishmanial activity of bovine serum albumin (BSA) nanoparticles containing amphotericin B (AmB) against Leishmania amazonensis. The antiproliferative activity against promastigotes and amastigotes was assessed and the cytotoxicity was determined and compared to commercial AmB-deoxycholate (AmB-D). In vivo antileishmania activity was evaluated in murine cutaneous leishmaniasis model. BSA nanoparticles showed spherical shape, mean size about 180 nm, zeta potential of ≈ -45 mV and AmB encapsulation efficiency >95%. AmB-D was effective in promastigote and amastigote forms, while AmB-loaded BSA nanoparticles were more effective against amastigotes than promastigotes. AmB-D was more effective than AmB-loaded BSA nanoparticles in both forms, however, the lowest cytotoxicity against macrophages was achieved by AmB-nanoparticles. BALB/c mice treated with AmB-D or AmB-loaded BSA nanoparticles showed a significant decrease in the lesion thickness at the infected footpad. Histopathological analysis after 3 weeks of treatment revealed AmB-D-related toxicity in heart, spleen, lung, liver and kidneys, while treatment with AmB-loaded BSA nanoparticles did not reveal tissue toxicity. The antileishmanial efficacy and the reduced toxicity become BSA nanoparticles containing AmB a potential candidate for treating cutaneous leishmaniasis.