Thein vitroantiplasmodial activity ofAmbrosia tenuifoliaorganic extract and its isolated sesquiterpene lactones, psilostachyin and peruvin, has been evaluated againstPlasmodium falciparumF32 and W2 strains. The cytotoxicity of both compounds was determined on lymphoid cells, and their corresponding selectivity indexes (SIs) were calculated. Peruvin was the most active compound on F32 strain ofP. falciparumwith a 50% inhibitory concentration value (IC50) of 0.3 μg/mL (1.1 μM) whereas psilostachyin showed activity on both strains (IC50= 0.6 (2.1 μM) and 1.8 μg/mL (6.4 μM)). Fifty percent cytotoxic concentration (CC50) values (48 h) were 6.8 μg/mL (24.3 μM) and 10.0 μg/mL (37.9 μM) for psilostachyin and peruvin, respectively.