Batch studies were conducted to evaluate the impact of biosurfactant (Rhamnolipid R1) on microbial utilization of radiolabeled 4,4′ chlorobiphenyl (4,4′CB). Production of 14CO2, after addition of an acclimated bacterial culture (Alcaligenes eutrophus), was monitored to determine the extent and rate of mineralization. Bioavailability of non-aqueous and soil-bound phases upon biosurfactant treatment was evaluated. Addition of Rhamnolipid R1 elevated both the rate and extent of 4,4′CB mineralization; however, enhancement was pronounced only at biosurfactant concentrations above the critical micelle concentration (CMC). For a biosurfactant concentration of 4.0 g/L, the average mineralization rate of 4,4′CB was 45 times that measured in controls (no biosurfactant). The extent of 4,4′CB mineralization in the presence of 4.0 g/L biosurfactant increased by a factor of 213 over biotic controls. Elevated mineralization was due, in part, to aqueous solubility enhancement of the test compound in the presence of biosurfactant. The total mass of 4,4′CB mineralized in biosurfactant solutions exceeded the total initial mass of 4,4′CB dissolved in biosurfactant free solutions, which confirmed that micellized 4,4′CB was biodegradable. Co-mineralization of biosurfactant was at a minimum during periods of high 4,4′CB mineralization. The results of this research provide clear evidence that biosurfactant addition followed by pure culture biological treatment is a promising approach for treatment of non-aqueous phase and soil-bound PCBs.