The current Chagas disease treatment is based on two drugs, nifurtimox and benznidazole, which is considered unsatisfactory, not only because of the narrow therapeutic range but also because of the associated toxicity. Natural products are considered an important source of biologically active compounds against various infectious organisms. Numerous Piper species are used in traditional medicine to treat parasitic diseases. In this paper, we study the activity of extracts and fractions obtained from Piper jericoense plant against epimastigote, trypomastigote and amastigote forms of Trypanosoma cruzi. In addition, we evaluated the cytotoxic, mutagenic and genotoxic activities of the F4 fraction obtained from one of the more promising extracts. We obtained four extracts, one of which presented low toxicity and high trypanocidal activity. This extract was separated into eight fractions, and the F4 fraction presented better results than the other extracts and had a higher selectivity index than the reference drug, benznidazole. This fraction was not cytotoxic, mutagenic or genotoxic.