This study investigated the efficiency of rhamnolipid biosurfactant and synthetic surfactant mixtures for improving the interfacial activity of the surfactant system against several light non-aqueous-phase liquids (LNAPLs). Since the rhamnolipid biosurfactant proved to be relatively hydrophilic, we hypothesized that mixtures of rhamnolipid biosurfactants with more hydrophobic synthetic surfactants would produce lower interfacial tensions (IFTs) than an individual rhamnolipid biosurfactant. The minimum IFT observed for rhamnolipid alone and toluene (0.03mN/m) was one order of magnitude lower than for hexane, decane, and hexadecane, demonstrating the relatively hydrophilic nature of the rhamnolipid. The low IFTs even at the low surfactant concentration used suggest mobilization as the dominant oil-removal mechanism versus supersolubilization. The critical micelle concentration (CMC) and critical microemulsion concentration (CmicroC) of the rhamnolipid were found to be 0.001w/w% (0.019mM) and 0.01w/w% (0.19mM), respectively. Three alkyl propoxylated (PO) sulfate synthetic surfactants were individually mixed with the rhamnolipid. As the hydrophobicity of the surfactant mixture approached that of the hydrocarbon, IFT values decreased by one to two orders of magnitude below that achieved with individual surfactants. This work shows that the rhamnolipid has excellent phase behavior at low concentrations and can be used in surfactant mixtures to achieve the low IFT values needed for environmental remediation, enhanced oil recovery (EOR), and other applications.