Protozoans of the genus Cryptosporidium are the causative agent of the gastrointestinal disease, cryptosporidiosis, which can be fatal in immunocompromised individuals. Cryptosporidium hominis (C. hominis) bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in the folate biosynthesis pathway and a molecular target for inhibitor design. Previous studies have demonstrated the importance of the ChTS-DHFR linker region "crossover helix" to the enzymatic activity and stability of the ChDHFR domain. We conducted a virtual screen of a novel non-active site pocket located at the interface of the ChDHFR domain and crossover helix. From this screen we have identified and characterized a noncompetitive inhibitor, compound 15, a substituted diphenyl thiourea. Through subsequent structure activity relationship studies, we have identified a time-dependent inhibitor lead, compound 15D17, a thiol-substituted 2-hydroxy-N-phenylbenzamide, which is selective for ChTS-DHFR, and whose effects appear to be mediated by covalent bond formation with a non-catalytic cysteine residue adjacent to the non-active site pocket.