Zequanox® is a biopesticide registered by the U.S. Environmental Protection Agency (USEPA) and the Canadian Pest Management Regulatory Agency for controlling dreissenid mussels with demonstrated selective toxicity. However, some research has indicated that Zequanox may impact the body condition and survival of some non-target species. We assessed avoidance behaviors of two species of cold-, cool-, and warm-water fishes to Zequanox at the maximum concentration allowed by the USEPA label (100 mg/L as active ingredient). Naïve, juvenile fish (n = 30 per species) were individually observed in a two-flume choice tank through which Zequanox-treated and untreated water simultaneously flowed in an unobstructed arena. Individual fish were observed during an untreated control period (20 min) and two Zequanox-exposure periods (20 min each). Treatment was alternated between arena sides to account for potential side bias in the test subjects. Positional data were collected and tabulated in real time with EthoVision® XT software. Zequanox concentrations and water quality properties (pH, dissolved oxygen, temperature, and specific conductance) were monitored during each trial. Analysis of treatment response was performed using a contrast within linear mixed-effects models. Our results indicate that Brook Trout, Lake Trout, and Bluegill avoided Zequanox-treated water, Yellow Perch were indifferent to Zequanox-treated water, and Lake Sturgeon and Fathead Minnow were attracted to Zequanox-treated water. These results combined with existing species sensitivity literature may help inform resource managers of potential treatment-related risks.