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ABSTRACT Francisella tularensis is a highly infectious Gram-negative bacterial pathogen capable of animal-to-human transmission. Due to its remarkable pathogenicity and potential for widespread public health impact, F. tularensis is classified as a high-priority pathogen by the Centers for Disease Control and Prevention. The historical development of Francisella as a bioweapon, coupled with the ease of engineering antibiotic-resistant strains, has spurred interest in the discovery of novel antibiotics. A library of drug-like compounds was screened for their ability to inhibit the growth of Francisella novicida, a biosafety level-2 surrogate for F. tularensis. Tolfenpyrad, a pesticide without previously identified antibacterial activity, was among the most potent inhibitors. Tolfenpyrad appears to uniquely target Francisella, as related species were sensitive, whereas bacteria in other genera were resistant. To identify Francisella pathways conferring sensitivity to tolfenpyrad, whole-genome sequencing of tolfenpyrad-resistant isolates was performed. Point mutations in two genes conferred resistance to osrR, a transcriptional regulator, and nuoM, a subunit of the electron transport chain. OsrR point mutants displayed attenuated growth in interferon gamma (IFN-γ)-stimulated macrophages but not in untreated macrophages, indicating that OsrR plays a role in resistance to IFN-γ-stimulated clearance of intracellular bacteria. OsrR point mutants are hypersensitive to reactive oxygen species, suggesting a mechanism by which they are rapidly cleared from IFN-γ-treated macrophages. This work identifies a novel Francisella-specific antibiotic activity and the metabolic pathways targeted. Moreover, we provide amino acid-level details of a Francisella protein that governs sensitivity to free radicals. IMPORTANCE Francisella species are highly pathogenic bacteria that pose a threat to global health security. These bacteria can be made resistant to antibiotics through facile methods, and we lack a safe and protective vaccine. Given their history of development as bioweapons, new treatment options must be developed to bolster public health preparedness. Here, we report that tolfenpyrad, a pesticide that is currently in use worldwide, effectively inhibits the growth of Francisella. This drug has an extensive history of use and a plethora of safety and toxicity data, making it a good candidate for development as an antibiotic. We identified mutations in Francisella novicida that confer resistance to tolfenpyrad and characterized a transcriptional regulator that is required for sensitivity to both tolfenpyrad and reactive oxygen species.