Background Among the tropical parasitic diseases, those caused by protozoans are considered a challenge to public health, being represented by leishmaniasis and Chagas disease. In view of the low effectiveness and toxicity of the current therapy, animal venoms such as amphibian secretions have been used as a promising source of new drug prototypes. The present work aimed to achieve bioguided fractionation of metabolites present in a cutaneous secretion of the caecilian Siphonops annulatus (Amphibia: Gymnophiona: Siphonopidae) with antileishmanial and antitrypanosomal activity.Methods Through liquid-liquid partition and chromatographic techniques, the secretion was fractionated using bioguided assays. The 50% inhibitory concentration (IC50) of the main fraction (SaFr1) was studied against Leishmania (L.) infantumpromastigotes and intracellular amastigotes, trypomastigotes ofTrypanosoma cruzi and mammalian cells; viability was detected by the colorimetric MTT assay. By using a spectrofluorimetric assay with the probe SYTOX® Green and transmission electron microscopy (TEM), we also investigated the potential damage caused by SaFr1 in the plasma membrane and mitochondria of Leishmania.Results The bioguided assay enabled isolation of a highly purified fraction (SaFr1) with an IC50 of 0.065 μg/mL against promastigotes and 2.75 μg/mL against trypomastigotes. Due to its high toxicity to peritoneal macrophages, SaFr1 showed no selectivity towards the intracellular forms ofLeishmania. Ultrastructural studies withLeishmania demonstrated severe mitochondrial damage and the formation of large cytoplasmic vacuoles, leading to the parasite's death within a few hours. Nevertheless, it caused no alteration in the plasma membrane permeability as detected by the fluorescent probe and TEM.Conclusions The present study demonstrated for the first time the antiparasitic activity of the skin secretion of the caecilian S. annulatus againstLeishmania and T. cruzi, confirming that skin secretions of these amphibians, similarly to those of anurans and salamanders, are also potential tools for the development of new drug candidates against neglected diseases.(AU)