The risk of lampricide applications (such as 4-nitro-3-[trifluoromethyl]phenol [TFM]) to nontarget fauna continues to be a concern within the Great Lakes Fishery Commission Sea Lamprey Control Program, especially among imperiled aquatic species—such as native freshwater mussels. The Grand River (Ohio, USA) is routinely treated for larval sea lampreys (Petromyzon marinus), and this river contains populations of the federally threatened mussel Obovaria subrotunda. Given this spatial overlap, information on the sensitivity of O. subrotunda to TFM is needed. Our objectives were to assess the toxicity of TFM to (1) adult Obovaria olivaria (a surrogate for O. subrotunda), (2) glochidial larvae of O. olivaria and O. subrotunda, (3) juveniles of O. olivaria and O. subrotunda, and (4) adult Percina maculata (host for O. subrotunda glochidia). In acute toxicity tests, TFM was not toxic to glochidia and adult mussels at exposure concentrations that exceed typical treatment rates. Although significant dose–response relationships were observed in hosts and juveniles, survival was ≥95% (Percina maculata), ≥93% (O. olivaria), and ≥74% (O. subrotunda) at typical treatment rates. However, the steep slope of these dose–response relationships indicates that an approximately 20% difference in the treatment level can result in nearly an order of magnitude difference in survival. Collectively, these data indicate that routine sea lamprey control operations are unlikely to acutely affect these species or their host. However, given that many mussel species are long-lived (30–100 years), the risks posed by lampricide treatments in the Great Lakes would be further informed by research on the potential long-term effects of lampricides on imperiled species. Environ Toxicol Chem 2024;00:1–8. Published 2024. This article is a U.S. Government work and is in the public domain in the USA.