Future modifications of fuels should include evaluation of the proposed constituents for their potential to damage environmental resources such as the subsurface environment. Batch and column experiments were designed to simulate biofuel spills in the subsurface environment and to evaluate the sorption and desorption behavior of target fuel constituents (i.e., monoaromatic and polyaromatic hydrocarbons) in soil. The extent and reversibility of the sorption of aromatic biofuel constituents onto soil were determined. When the ethanol content in ethanol-blended gasoline exceeded 25%, enhanced desorption of the aromatic constituents to water was observed. However, when biodiesel was added to diesel fuel, the sorption of target compounds was not affected. In addition, when the organic carbon content of the soil was higher, the desorption of target compounds into water was lower. The empirical relationships between the organic-carbon normalized sorption coefficient (Koc) and water solubility and between Koc and the octanol-water partition coefficient (Kow) were established. Column experiments were carried out for the comparison of column effluent concentration/mass from biofuel-contaminated soil. The dissolution of target components depended on chemical properties such as the hydrophobicity and total mass of biofuel. This study provides a basis for predicting the fate and transport of hydrophobic organic compounds in the event of a biofuel spill. The spill scenarios generated can assist in the assessment of biofuel-contaminated sites.