The necessity of drug combinations to treat leishmaniasis came to the surface mainly because of the toxicity of current treatments and the emergence of resistant strains. The calpain inhibitor MDL28170 has previously shown anti-Leishmania activity, therefore its use in association with standard drugs could provide a new alternative for the treatment strategy against leishmaniasis. In this study, we analyzed the potential of the combination of MDL28170 and the antileishmanial drug amphotericin B against Leishmania amazonensis and Leishmania chagasi. The compounds were tested in the combination of the ½ × IC50 value of MDL28170 plus the ¼ × IC50 value of amphotericin B, which led to an increment in the anti-promastigote activity when compared to the single drug treatments. This drug association revealed several and severe morphophysiological changes on parasite cells, such as loss of plasma membrane integrity, reduced size of flagellum, and depolarization of mitochondrial membrane potential besides increased reactive oxygen species production. In addition, the combination of both drugs had a deleterious effect on the Leishmania–macrophage interaction, reflecting in a significant anti-amastigote action, which achieved a reduction of 50% in the association index. These results indicate that the combination treatment proposed here may represent a new alternative for leishmaniasis chemotherapy.