Leishmaniasis is a neglected tropical disease that affects millions of people worldwide. Current therapies mainly rely on antimonial drugs that are inadequate because of their high toxicity and increased drug resistance. An urgent need exists to discover new, more effective, more affordable, and more target-specific drugs. Pathways that are associated with apoptosis-like cell death have been identified in unicellular eukaryotes, including protozoan parasites. In the present study, we studied the mechanism of cell death that is induced by A3K2A3 against L. amazonensis. A3K2A3 is a dibenzylideneacetone that has an acyclic dienone that is attached to aryl groups in both β-positions, which is similar to curcuminoids and chalcone structures. This compound was previously shown to be safe with regard to cytotoxicity and active against the parasite. Biochemical and morphological approaches were used in the present study. The results suggested that A3K2A3 caused mitochondrial dysfunction in L. amazonensis promastigotes, leading to mechanisms of cell death that share some common phenotypic features with metazoan apoptosis, such as an increase in reactive oxygen species production, a decrease in the adenosine triphosphate ratio, phosphatidylserine exposure, a decrease in cell volume, caspase production, and DNA fragmentation. Altogether, these findings indicate that apoptosis can indeed be triggered by chemotherapeutic agents.