Zequanox® is a commercial formulation of the killed bacterium, Pseudomonas fluorescens (strain CL145A), that was developed to control dreissenid mussels. In 2014, Zequanox became the second product registered by the United States Environmental Protection Agency (USEPA) for use in open water environments as a molluscicide. Previous nontarget studies demonstrated the safety and selectivity of P. fluorescens CL154A, but the database on the toxicity of the formulation (Zequanox) is limited for macroinvertebrate taxa and exposure conditions. We evaluated the safety of Zequanox to the amphipod Gammarus lacustris lacustris, and nymphs of the burrowing mayfly, Hexagenia spp. at the maximum approved concentration (100 mg/L active ingredient, A.I.) and exposure duration (8 h). Survival of animals was assessed after 8 h of exposure and again at 24 and 96 h post-exposure. Histopathology of the digestive tract of control and treated animals was compared at 96 h post-exposure. The results showed no significant effect of Zequanox on survival of either species. Survival of G. lacustris exceeded 85% in all concentrations at all three sampling time points. Survival of Hexagenia spp. ranged from 71% (control) to 91% at 8 h, 89–93% at 24 h post-exposure, and 70–73% at 96 h post-exposure across all treatments. We saw no evidence of pathology in the visceral organs of treated animals. Our results indicate that application of Zequanox at the maximum approved concentration and exposure duration did not cause significant mortality or treatment-related histopathological changes to G. lacustris and Hexagenia spp.