The degradation of crude and weathered crude oil following the application of crude and calcium-alginate encapsulated ligninolytic enzymes was studied using in situ microcosms. Changes in the chemical composition of the oil were monitored in crude enzyme extracts, as well as a sediment matrix, for as long as 70 days. Compound specific effects of ligninolytic enzymes applied to the sediments were observed over time through changes in concentration of total petroleum hydrocarbons (TPH), polycyclic aromatic hydrocarbons (PAHs) and fractions of saturates, aromatics, resins and asphaltenes (SARA). As the oil weathered, most TPH and PAH fractions showed a rapid decrease in concentration. As sediment oil concentrations decreased following treatment with ligninolytic enzymes, the microbial population was enriched with hydrocarbon-degrading species. This trend demonstrates that the oil fractions initially not bioavailable for microbial degradation, were subsequently released to the sediment via catalytic conversion with laccase and manganese peroxidase, and the oil continues to be biodegraded by microbial populations.