The pharmacokinetics, bioavailability, and disposition of oxolinic acid (OA), a quinolone antibacterial drug, were examined in a warmwater (channel catfish (Ictalurus punctatus)) and coldwater fish species (rainbow trout (Oncorhynchus mykiss)). Studies were performed at 24 and 14 °C for catfish and at 14 °C for trout to compare temperature- and species-related differences. Analysis of OA pharmacokinetics when determined by HPLC provided elimination half-life [Formula: see text], volume of distribution (Vss), and clearance (Clb) estimates for 24 °C catfish, 14 °C catfish, and 14 °C trout of 40.9, 69.3, and 81.3 h, 939, 880, and 1817 mL/kg, and 16.3, 8.9, and 16.9 mL∙kg∙h−1, respectively. Following oral administration, OA plasma concentrations peaked between 8 and 24 h for all treatments; however, 14 °C trout and 14 °C catfish sustained peak concentrations for a longer duration than 24 °C catfish. Oral OA bioavailabilities were 56.0, 91.8, and 90.7% for 24 °C catfish, 14 °C catfish, and 14 °C trout, respectively. OA distribution data for muscle of 14 °C catfish demonstrated an inordinately high peak concentration and delayed time to peak relative to other treatments. Elimination half-lives of OA in muscle were 33.1, 54.3, and 141 h for 24 °C catfish, 14 °C catfish, and 14 °C trout, respectively.