Background: Several species of Aspidosperma (Apocynaceae) are used as treatments for human diseases in the tropics. Aspidosperma olivaceum, which is used to treat fevers in some regions of Brazil, contains the monoterpenoid indole alkaloids (MIAs) aspidoscarpine, uleine, apparicine, and N-methyl-tetrahydrolivacine. Using bio-guided fractionation and cytotoxicity testing in a human hepatoma cell line, several plant fractions and compounds purified from the bark and leaves of the plant were characterized for specific therapeutic activity (and selectivity index, SI) in vitro against the blood forms of Plasmodium falciparum. Methods: The activity of A. olivaceum extracts, fractions, and isolated compounds was evaluated against chloroquine (CQ)-resistant P. falciparum blood parasites by in vitro testing with radiolabelled [3H]-hypoxanthine and a monoclonal anti-histidine-rich protein (HRPII) antibody. The cytotoxicity of these fractions and compounds was evaluated in a human hepatoma cell line using a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay, and the SI was calculated as the ratio between the toxicity and activity. Two leaf fractions were tested in mice with Plasmodium berghei. Results: All six fractions from the bark and leaf extracts were active in vitro at low doses (IC50 < 5.0 μg/mL) using the anti-HRPII test, and only two (the neutral and basic bark fractions) were toxic to a human cell line (HepG2). The most promising fractions were the crude leaf extract and its basic residue, which had SIs above 50. Among the four pure compounds evaluated, aspidoscarpine in the bark and leaf extracts showed the highest SI at 56; this compound, therefore, represents a possible anti-malarial drug that requires further study. The acidic leaf fraction administered by gavage to mice with blood-induced malaria was also active. Conclusion: Using a bio-monitoring approach, it was possible to attribute the anti-P. falciparum activity of A. olivaceum to aspidoscarpine and, to a lesser extent, N-methyl-tetrahydrolivacine; other isolated MIA molecules were active but had lower SIs due to their higher toxicities. These results stood in contrast to previous work in which the anti-malarial activity of other Aspidosperma species was attributed to uleine.