The objective of the present study was to compare the effect of rhamnolipids on the microbial biomass content and the activity of dehydrogenases (DHA), acid phosphatase (ACP), alkaline phosphatase (ALP), and urease (URE) in soil contaminated with two types of coal tar creosote: type C and type GX-Plus. The experiment was carried out on samples of sandy clay loam under laboratory conditions. Coal tar creosote was added to soil samples at a dose of 0 and 10 g·kg−1 DM, along with rhamnolipids at a dose of 0, 10, 100, and 1000 mg·kg−1 DM. The humidity of the samples was brought to 60% maximum water holding capacity, and the samples were incubated at 20°C. Microbial and biochemical parameters were determined on days 1, 7, 21, and 63. The obtained results demonstrated that the addition of rhamnolipids did not result in any significant changes in the activity of the determined parameters in the uncontaminated soil. However, it was observed that the application of these biosurfactants, particularly at the dose of 1000 mg·kg−1 DM, largely decreased the effect of coal tar creosote on the determined parameters. Moreover, the microbial biomass and the activity of ALP and URE were found to be the best indicator of bioremediation of soil contaminated with coal tar creosote.