We conducted concurrent 8-week feeding trials with fathead minnow (FHM) (Pimephales promelas) fed diets with 0 or 2% dairy–yeast prebiotic in recirculating systems with low (LOW, 35 mg CaCO3 per L water) or moderate (MOD, 141 mg CaCO3 per L water) hardness to determine the effects of the prebiotic and water hardness on general performance, whole-body ash and mineral concentrations, and composition of the gastrointestinal (GI) microflora.