Leishmaniasis is caused by parasites of the genus Leishmania. Recent reports about leishmaniasis show a few number of drugs available, indicating the necessity of new drugs. In this study, the ethanol extract and fractions of Pityrogramma calomelanos (L.) link. (Pteridaceae) were assayed to verify the cytotoxicity and in vitro leishmanicidal activity against promastigote forms of Leishmania brasiliensis. The cytotoxic assay was performed using fibroblasts NCTC929. The studies indicated a leishmanicidal effect of the ethanol extract and the ethyl-acetate fraction. However, a high cytotoxic effect was observed. The hexane and methanol fractions did not show leishmanicidal activity, nor cytotoxic effect. The phytochemical screening detected the presence of alkaloids, a class of secondary metabolites with a known leishmanicidal activity. This is the first study reporting an anti-Leishmania and cytotoxic effect of P. calomelanos, being an interesting approach in the search for drugs against this disease.