Malaria is responsible for 3 million deaths annually. Antimalarial drug resistance is widespread, and few novel, well-defined targets exist. A robotic high throughput screen (HTS) was performed using 4000 small molecules from a natural compound (Spectrum), pharmacologically active (Lopac), and Food and Drug Administration (FDA) approved drug library (Prestwick) for competitive inhibition of the ATP-binding (GHKL) domain of Plasmodium falciparum (Pf) Hsp90, a highly conserved chaperone. Hits were further screened for specificity based on differential inhibition of PfHsp90 in comparison to human (Hs) Hsp90. PfHsp90-specific inhibitors showed 50% inhibitory concentrations (IC(50)) in the nanomolar range when tested using a cell-based antimalarial validation assay. Three hits, identified as selective PfHsp90 inhibitors in the HTS, also demonstrated synergistic activity in the presence of the known antimalarial drug chloroquine. These data support PfHsp90 as a specific antimalarial target with potential for synergy with known antimalarials.