The in vitro leishmanicidal effect of (3S)-16,17-didehydrofalcarinol (1) isolated from Tridax procumbens whole plant against Leishmania mexicana, the causative agent of cutaneous leishmaniasis (chiclero's ulcer) in the New World, was investigated. This oxylipin showed significant in vitro activity against promastigotes and intracellular amastigotes of L. mexicana. Its inhibitory effect on amastigotes was not due to activation of NO in recombinant gamma-interferon-stimulated macrophages, since the production of NO was decreased in presence of the oxylipin. This is the first report on the leishmanicidal activity against the intracellular stage (amastigote) of the oxylipin (3S)-16,17-didehydrofalcarinol.