Chagas disease is a tropical disease caused by the parasite Trypanosoma cruzi (T cruzi), which is endemic to Central and South America. Approximately 13 million people are infected with the parasite, and 25–30% of infected patients suffer from irreversible damage to the heart and digestive tract resulting in disability and death within 20 years of infection. Few treatments are available with limited effectiveness only against the early (acute) stages of disease, significant toxicity, and widespread drug resistance. We report the outcome of a high-throughput chemical library screen to identify novel, nontoxic, small-molecule inhibitors of T cruzi, which will aid the development of more potent and selective therapies for both the acute and chronic stages of Chagas disease. Of the 303,224-screened compounds, 35 compounds were chosen based on their selectivity, potency, and chemical tractability. Of those, 27 dry powder-validated compounds were retested in the primary screen, secondary assays, and an orthogonal screen. Three scaffolds were prioritized to identify potential probes. One of these compounds, ML157 (CID-16190867) displayed greater than 100-fold selective inhibition of T cruzi as confirmed in immunofluorescent imaging assays and is inactive against host cells at the highest tested dose. Our results show that ML157 has a drug-like structure with no obvious chemical liabilities, excellent solubility, and stability in aqueous conditions. This new probe should be very useful in future cell-based investigations and in vivo studies of T cruzi inhibition.