Herein, we describe the hit optimization of a novel diarylthioether chemical class found to be active against Trypanosoma cruzi; the parasite responsible for Chagas disease. The hit compound was discovered through a whole-cell phenotypic screen and as such, the mechanism of action for this chemical class is unknown. Our investigations led to clear structure-activity relationships and the discovery of several analogues with high in vitro potency. Furthermore, we observed excellent activity during acute in vivo efficacy studies in mice infected with transgenic T. cruzi. These diarylthioether compounds represent a promising new chemotype for Chagas disease drug discovery and merit further development to increase oral exposure without increasing toxicity.