A number of methods exist for removing petroleum hydrocarbons from water to lower the risk to the environment. Firefighting training generates a unique case of immiscible-phase hydrocarbons with intermittent flow, high total petroleum hydrocarbon (TPH) concentrations, and strict regulations. The available treatment options for this contaminated water have a number of disadvantages that include: expense, waste disposal, fluctuating effectiveness, and intensive manual maintenance that make it unsuitable for firefighting training centers. Thus, a bio-separator was designed for remediating the impacts of immiscible-phase hydrocarbon using horizontal flow through a layer of mulch and a layer of aggregate in series. Free-phase hydrocarbon is retained in a fuel storage reservoir while remediated water flows out of an inverted siphon. This research 1) completes a comprehensive literature review on methods for removing petroleum hydrocarbons from runoff and the performance of wood mulch for stormwater pollutant removal, 2) elucidates on the physical and biological removal processes of the bio-separator, and 3) investigates and tests design options for the bio-separator. Using the results from the tests, multiple regression analysis was performed and predictive equations for immiscible-phase hydrocarbon breakthrough time and concentration reduction were found. A case study bio-separator was designed for the Oklahoma State University (OSU) Fire Service Training Center. The results suggest that further work testing the bio-separator at a larger scale is needed before widespread implementation.