Anthrax lethal factor (LF) is a key virulence factor of anthrax lethal toxin. We screened a chemolibrary of 10,000 drug-like molecules for their ability to inhibit LF and identified 18 novel small molecules with potent LF inhibitory activity. Three additional LF inhibitors were identified through further structure-activity relationship (SAR) analysis. All 21 compounds inhibited LF with an IC50 range of 0.8 to 11 muM, utilizing mixed-mode competitive inhibition. An evaluation of inhibitory activity against a range of unrelated proteases showed relatively high specificity for LF. Furthermore, pharmacophore modeling of these compounds showed a high degree of similarity to the model published by Panchal et al. (Nat. Struct. Mol. Biol. 2004, 11, 67-72), indicating that the conformational features of these inhibitors are structurally compatible with the steric constraints of the substrate-binding pocket. These novel LF inhibitors and the structural scaffolds identified as important for inhibitory activity represent promising leads to pursue for further LF inhibitor development.