Zequanox® is a commercial formulation of the killed bacterium, Pseudomonas fluorescens (strain CL145A) that has USEPA approval for use in open water to kill dreissenid mussels. Previous nontarget studies demonstrated the safety and selectivity of the product, but the database is limited for macroinvertebrate taxa and exposure conditions. We evaluated the safety of the product to two high value macroinvertebrates, the amphipod Gammarus lacustris lacutris, and nymphs of the burrowing mayfly, Hexagenia spp., at the maximum approved concentration (100 mg/L A.I.) and exposure duration (8 h). Survival of animals was measured at the end of 8 h exposure and at 24 h post-exposure and 96 h post-exposure. Additionally, histological changes in the digestive tract of both species were compared with controls at 96 h post-exposure. The results showed no 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% (50 mg/L) at 8 h, 89-93% at 24 h post-exposure and 70-73% at 96 post-exposure h across all treatments. We also 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 does not cause acute mortality or pathology to G. lacustris and Hexagenia spp.