Analogues of the natural antibiotic thiolactomycin (TLM), an inhibitor of the condensing reactions of type II fatty acid synthase, were synthesized and evaluated for their ability to inhibit the growth of the malaria parasite Plasmodium falciparum. Alkylation of the C4 hydroxyl group led to the most significant increase in growth inhibition (over a 100-fold increase in activity compared to TLM). To investigate the mode of action, the P. falciparum KASIII enzyme was produced for inhibitor assay. A number of TLM derivatives were identified that showed improved inhibition of this enzyme compared to TLM. Structure-activity relationships for enzyme inhibition were identified for some series of TLM analogues, and these also showed weak correlation with inhibition of parasite growth, but this did not hold for other series. On the basis of the lack of a clear correlation between inhibition of pfKASIII activity and parasite growth, we conclude that pfKASIII is not the primary target of TLM analogues. Some of the analogues also inhibited the growth of the parasitic protozoa Trypanosoma cruzi, T. brucei, and Leishmania donovani.