This dataset includes the behavioral fish data needed to assess the responses of Upper Mississippi River fishes to an acoustic stimulus (i.e., playback of a 100 hp boat motor). Native ostariophysians (bigmouth buffalo Ictiobus cyprinellus, channel catfish Ictalurus punctatus, and fathead minnow Pimephales promelas), invasive ostariophysians (bighead carp Hypophthalmichthys nobilis, common carp Cyprinus carpio, grass carp Ctenopharyngodon idella, and silver carp Hypophthalmichthys molitrix), and native non-ostariophysians (American eel Anguilla rostrata, gizzard shad Dorosoma cepedianum, hybrid striped bass Morone saxatilis, lake sturgeon Acipenser fulvescens, and paddlefish Polyodon spathula) were exposed to a playback of a 100 hp boat motor sound in indoor tanks, and their behavior was monitored for negative phonotaxis and changes in behavior.