This study evaluated the pharmacokinetics of florfenicol in the white-spotted bamboo shark (Chiloscyllium plagiosum). In addition to the pharmacokinetics, the potential application for treatment of bacterial meningitis was explored. A pilot study was used to compare doses of 30, 40, and 50 mg/kg i.m. Following that study, 10 adult sharks were administered a single i.m. dose of florfenicol at 40 mg/kg. Plasma and cerebrospinal fluid were collected and analyzed for florfenicol by a sensitive and specific high-pressure liquid chromatographic method. Pharmacokinetic analysis was performed using both non-compartmental and compartmental techniques. The absorption produced an average peak at 54 (±19) hr from the i.m. site of administration, and the half-life was prolonged, averaging 269.79 hr (±135.87). Florfenicol plasma concentrations peaked at an average of 11.85 μg/ml (±1.45) and were maintained above our target minimum inhibitory concentration of 4–8 μg/ml for at least 120 hr. Cerebrospinal fluid concentrations peaked at an estimated 9 μg/ml around 48 hr, surpassing the target minimum inhibitory concentration for at least 72 hr.