AbstractNatural products represent a rich source of new chemical entities for the development of drugs for neglected diseases. Leishmaniasis still afflicts the poorest populations in the world, with 12 million cases worldwide. This work analysed crude extracts and fractions from the fruits of Cassia fistula against the most dramatic and fatal disease form of leishmaniasis, the visceral form (VL). Hexane extract from the fruits showed significant antileishmanial activity against the promastigote form of Leishmania L. chagasi. The bioguided fractionation resulted in the isolation of a sterol, clerosterol, which was further analysed in different models. Promastigotes presented an inhibitory concentration 50% (IC50) of 10.03 µg/mL and intracellular amastigotes demonstrated high susceptibility, with an IC50 of 18.10 µg/mL. Mammalian cytotoxicity was evaluated and it was demonstrated that clerosterol was 3.6‐fold less toxic than the standard drug pentamidine. No antifungal activity of the isolated clerosterol was found. Future studies of the extracted compounds of Cassia fistula could be a useful tool for the development of new therapeutic agents for VL. Copyright © 2007 John Wiley & Sons, Ltd.