Cruzain is a key cysteine protease that is essential for the survival and replication of Trypanosoma cruzi (T. cruzi), a protozoan parasite that is the causative agent of Chagas disease. Inhibition of cruzain has been validated as a viable strategy for the development of small molecule therapeutics for Chagas disease. To date, reports of small molecule cruzain inhibitors have been primarily of those which contain an electrophilic warhead and act by irreversible, covalent modification of the enzyme. As such, we sought to discover novel reversible, non-covalent inhibitors of cruzain using a combination of docking, co-crystallization and high-throughput screening. In this report, we describe the discovery of ML217, which exhibits trypanocidal activity against the T. Cruzi parasite, while having minimal toxicity to the host cell. Moreover, ML217 represents the first reversible non-covalent inhibitor of cruzain to demonstrate efficacy in a Chagas disease mouse model.