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Small molecules have been shown to be potent and selective probes to understand cell physiology. Here, we show that imidazo[1,2-a]pyridines and imidazo[1,2-a]pyrimidines compose a class of compounds that target essential, conserved cellular processes. Using validated chemogenomic assays in Saccharomyces cerevisiae, we discovered that two closely related compounds, an imidazo[1,2-a]pyridine and -pyrimidine that differ by a single atom, have distinctly different mechanisms of action in vivo. 2-phenyl-3-nitroso-imidazo[1,2-a]pyridine was toxic to yeast strains with defects in electron transport and mitochondrial functions and caused mitochondrial fragmentation, suggesting that compound 13 acts by disrupting mitochondria. By contrast, 2-phenyl-3-nitroso-imidazo[1,2-a]pyrimidine acted as a DNA poison, causing damage to the nuclear DNA and inducing mutagenesis. We compared compound 15 to known chemotherapeutics and found resistance required intact DNA repair pathways. Thus, subtle changes in the structure of imidazo-pyridines and -pyrimidines dramatically alter both the intracellular targeting of these compounds and their effects in vivo. Of particular interest, these different modes of action were evident in experiments on human cells, suggesting that chemical–genetic profiles obtained in yeast are recapitulated in cultured cells, indicating that our observations in yeast can: (1) be leveraged to determine mechanism of action in mammalian cells and (2) suggest novel structure–activity relationships., Author Summary We have shown that chemical–genetic screening allows structure–activity studies of chemical compounds at a very high resolution. In analyzing the effects of closely related imidazo-pyridine and -pyrimidine compounds, we found two compounds that likely act as oxidizing agents, yet target different organelles. The imidazo-pyridine affected mitochondrial functions whereas the imidazo-pyrimidine caused nuclear DNA damage. Remarkably, the only difference between these two compounds is the presence of a nitrogen atom at position 8. Thus, in addition to demonstrating the potential for high resolution in chemical–genetic studies, our work suggests that subtle changes in compound chemistry can be exploited to target different intracellular compartments with very different biological effects. Finally, we show that chemical–genetic profiling in yeast can be used to infer mode of action in mammalian cells. The specificity of compound 15 in eliciting a nuclear DNA damage response in evolutionarily diverse eukaryotes suggests that it will be of great utility in studying the cellular response to nuclear oxidative damage.