Twigs of <i>Nectandra barbellata</i> were extracted using a solution of the ionic liquid 1-butyl-3-methylimidazolium bromide (BMImBr) in H<sub>2</sub>O, assisted by microwave (MAE). After successive chromatographic steps, one sesquiterpene, costic acid, and three new related lactones, <i>(R)-</i>3(7)-<i>Z</i>-3-hexadec-21-enylidene-5-(hydroxymethyl)tetrahydrofuran-2-one (<b>1</b>), <i>(R)-</i>3(7)-<i>Z</i>-3-hexadecylidene-5-(hydroxymethyl)tetrahydrofuran-2-one (<b>2</b>), and <i>(R)-</i>3(7)-<i>Z</i>-3-docosylidene-5-(hydroxymethyl)tetrahydrofuran-2-one (<b>3</b>), were isolated. After structural elucidation using IR, UV, HRESIMS, NMR, ECD, and VCD, compounds <b>1-3</b> were tested against trypomastigote forms of <i>Trypanosoma cruzi</i>. The mechanism of action of bioactive isolated compounds was studied using different fluorescent-based approaches to investigate alterations of the plasma membrane, permeability/electric potential (ΔΨ<sub>p</sub>), reactive oxygen species levels, mitochondria (electric membrane potential, ΔΨ<sub>m</sub>/ATP levels), Ca<sup>2+</sup> levels, and pH of the acidocalcisomes. In addition, in silico studies predicted no resemblance to pan assay interference compounds (PAINS).