Current therapies for Chagas disease and leishmaniasis have limited efficacy, significant toxicity, and suboptimal cure rates. In this context, natural products represent a powerful tool for the discovery and development of new agents to overcome these limitations. Here we report the synthesis, antikinetoplastid activity, and proposed mechanism of action of a unique class of withaferin A derivatives ( 2 - 19 ). These compounds showed a potent inhibitory effect on the proliferation of Trypanosoma cruzi epimastigotes (compounds 5 , 6 , 12 - 14 , 16 and 17 ) and Leishmania amazonensis promastigotes (compounds 4 - 6 , 9 , 12 - 14 , 16 and 17 ). Notably, compounds 14 and 16 emerged as the most promising compounds, exhibiting higher potency than the reference drug, against promastigotes and amastigotes stage of L. amazonensis , along with a high selectivity index on a normal eukaryotic cell line. Additionally, compounds 14 and 16 induced apoptosis-like programmed cell death on L. amazonensis , mediated by the disruption of ATP levels, mitochondrial membrane potential, and chromatin condensation, and energy depletion. These findings reinforce the therapeutic potential of withanolides, providing strong support for the continued development of withaferin A (WA) derivatives as potential antikinetoplastid agents.