Natural products are a promising source of new compounds with a wide spectrum of pharmacological properties, including antiprotozoal activities. Chagas disease, caused by the protozoan parasiteTrypanosoma cruzi, is one of several neglected tropical diseases with reduced options for treatment, which presents limitations such as toxicity and ineffectiveness in the chronic stage of the disease. Aiming to investigate the Brazilian flora for the discovery of new anti-T. cruzicompounds, the MeOH extract fromPorcelia macrocarpaR.E. Fries (Annonaceae) fruit peels displayed potent activity against trypomastigotes and intracellular amastigotes and was subjected to bioactivity-guided fractionation. Using different chromatographic steps, a fraction composed of a mixture of four new chemically related acetogenins was obtained. The compounds were characterized as (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-octadeca-13′,17′-dien-11′-inil)butanolide (1), (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-eicosa-13′,19′-dien-11′-inil)butanolide (2), (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-octadec-13′-en-11′-inil)butanolide (3), and (2S*,3R*,4R*)-3-hydroxy-4-methyl-2-(n-eicosa-13′-en-11′-inil)butanolide (4) by NMR analysis and UHPLC/ESI-HRMS data. The mixture of compounds1–4,displayed an EC50of 4.9 and 2.5 µg/mL against trypomastigote and amastigote forms ofT. cruzi, respectively, similar to the standard drug benznidazole (EC50of 4.8 and 1.4 µg/mL). Additionally, the mixture of compounds1–4displayed no mammalian toxicity for murine fibroblasts (CC50> 200 µg/mL), resulting in a SI > 40.8 and > 83.3 against trypomastigotes and amastigotes, respectively. Based on these results, the mechanism of action of this bioactive fraction was investigated. After a short-time incubation with the trypomastigotes, no alterations in the cell membrane permeability were observed. However, it was verified a decrease in the intracellular calcium of the parasites, without significant pH variations of the acidocalcisomes. The intracellular damages were followed by an upregulation of the reactive oxygen species and ATP, but no depolarization effects were observed in the mitochondrial membrane potential. These data suggest that the mixture of compounds1–4caused an irreversible oxidative stress in the parasites, leading to death. If adequately studied, these acetogenins can open new insights for the discovery of new routes of death inT. cruzi.