The identification of antiplasmodial lead compounds from the dry seeds of Piper guineense has been performed using a bioassay-directed protocol. Briefly, its methanolic seeds extract has been suspended in water and partitioned using n-hexane, dichloromethane, ethyl acetate and n-butanol to afford four main fractions A−D which were further submitted to antiplasmodial assay against Plasmodium falciparum 3D7. The bioassay results showed that its dichloromethane-soluble fraction has the highest potency with 31.8 % of the parasite P. falciparum 3D7 survival. The purification of the active fraction with successive column chromatography led to the isolation and identification of eleven compounds including six piperine-type amides (1−6), two lignans (7 and 8), one fatty acid (9), and two steroids (10 and 11). The antiplasmodial assay of compounds 1−8 against the two strains Pf 3D7 and Pf Dd2 showed that only the alkaloids 1−6 displayed moderate potencies with the highest IC50 values of 17.4 ± 0.7 µg/mL (for 6 against Pf 3D7) and 14.6 ± 0.3 µg/mL (for 2 against Pf Dd2). Furthermore, the cytotoxicity of the tested compounds against Vero cell lines showed that only compounds 5 and 6 displayed a cytotoxicity with CC50 values of 335.4 ± 7.6 µg/mL and 151.8 ± 2.4 µg/mL, respectively, while their selectivity index limits an acceptable range. The results support the use of the plant in folk medicine for handling malaria, and demonstrate that piperine-type amides are responsible for the antiplasmodial activity of P. guineense and are good leads in the development of safe and potent antimalarial drugs.