Background: Neglected Infectious Diseases control is an urgent need in endemic areas, but it’s not a priority in the commercial interests of the pharmaceutical industry. Chagas disease (caused by Trypanosoma cruzi) is the parasitic disease of greatest impact in Latin America. Medicinal plants continue to be an affordable front-line option in endemic areas. Thus, we aimed to advance in the pharmacological evaluation of four medicinal plants traditionally used in the Amazon against parasitic infections. Materials and Methods: The plants were collected in the Amazon region of Colombia. The dry plant material was submitted to water percolation extraction. Extracts were tested in vitro against Trypanosoma cruzi (epimastigotes), and cytotoxicity was assessed against HepG2 and MRC5 cells. Finally, the general profile of metabolites present in the extracts was studied by thin-layer chromatography. Results: In vitro, against T. cruzi, extracts of Ambelania duckei and Curarea toxicofera shows concentration- dependent inhibition (IC50 of 221+/-29 and 50+/-5 μg/mL respectively), comparable with benznidazole (IC50: 0.7 μg/mL); while Abuta grandifolia and Aspidosperma excelsum exhibited IC50’s > 500 μg/mL. All extracts showed no cytotoxicity against HepG2 and MRC5 cells. Yields of extraction were between 3.2 and 9.5% and preliminary phytochemical profile showed flavonoids and steroids in all extracts. Conclusion: Promising plants, traditionally used to treat other protozoan infections, could be assessed against T. cruzi. C. toxicofera exhibits good activity against epimastigotes of T. cruzi, being a species that can reasonably be considered for bioassay-guided antitrypanosomal fractionation.