ABSTRACT We tested the antituberculosis drug SQ109, which is currently in advanced clinical trials for the treatment of drug-susceptible and drug-resistant tuberculosis, for its in vitro activity against the trypanosomatid parasite Trypanosoma cruzi , the causative agent of Chagas disease. SQ109 was found to be a potent inhibitor of the trypomastigote form of the parasite, with a 50% inhibitory concentration (IC ₅₀ ) for cell killing of 50 ± 8 nM, but it had little effect (50% effective concentration [EC ₅₀ ], ∼80 μM) in a red blood cell hemolysis assay. It also inhibited extracellular epimastigotes (IC ₅₀ , 4.6 ± 1 μM) and the clinically relevant intracellular amastigotes (IC ₅₀ , ∼0.5 to 1 μM), with a selectivity index of ∼10 to 20. SQ109 caused major ultrastructural changes in all three life cycle forms, as observed by light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). It rapidly collapsed the inner mitochondrial membrane potential (Δψ _m ) in succinate-energized mitochondria, acting in the same manner as the uncoupler FCCP [carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone], and it caused the alkalinization of internal acidic compartments, effects that are likely to make major contributions to its mechanism of action. The compound also had activity against squalene synthase, binding to its active site; it inhibited sterol side-chain reduction and, in the amastigote assay, acted synergistically with the antifungal drug posaconazole, with a fractional inhibitory concentration index (FICI) of 0.48, but these effects are unlikely to account for the rapid effects seen on cell morphology and cell killing. SQ109 thus most likely acts, at least in part, by collapsing Δψ/ΔpH, one of the major mechanisms demonstrated previously for its action against Mycobacterium tuberculosis . Overall, the results suggest that SQ109, which is currently in advanced clinical trials for the treatment of drug-susceptible and drug-resistant tuberculosis, may also have potential as a drug lead against Chagas disease.