The risk of lampricide applications (such as 4-Nitro-3-(trifluoromethyl)phenol, TFM) to non-target 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) is routinely treated for larval sea lampreys (Petromyzon marinus) and this river contains populations of the Round Hickorynut mussel (Obovaria subrotunda) which was Federally listed in 2023. Given the spatial overlap between areas treated with TFM and extant populations of O. subrotunda, information on its sensitivity to TFM is needed. This dataset was used to evaluate the acute toxicity of TFM to O. olivaria and O. subrotunda, as well as Percina maculata (host for O. subrotunda glochidia). We found that in acute toxicity tests, the lampricide 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 juveniles and host fish, mortality was ≤5% (P. maculata), ≤7% (O. olivaria), and ≤26% (O. subrotunda) at typical treatment rates. However, the steep slope of these dose-response relationships indicate that a ~20% difference in the treatment level can result in nearly an order of magnitude difference in mortality. Collectively, these data indicate that routine sea lamprey control operations are unlikely to acutely affect these species or their host; however, given that mussels are long-lived (30–100 y), the risks posed by lampricide treatments in the Great Lakes would be further informed by research on the long-term effects of lampricides on imperiled species.