Biosurfactants are promising alternatives to chemical dispersants for combating marine oil spills; however, the impacts of biosurfactants on microbial community composition and oil biodegradation activities remain largely unknown. Here, we conducted a time-course microcosm experiment mimicking oil spill scenarios with surface seawater from the North Sea, amended with either the biosurfactant rhamnolipid or a dispersant (Corexit 9500 or Slickgone NS). Radioactive tracer assays to track hexadecane and naphthalene oxidation as well as bacterial production revealed the highest hydrocarbon oxidation rates and general microbial activities in the rhamnolipid-amended oil microcosms, followed by oil microcosms with Slickgone and Corexit. Impacts on the microbial community composition differed among treatments, and growth of oil-degrading Colwellia was stimulated remarkably in Corexit-amended oil and oil-only microcosms, while potential oil-degrading Oleispira were highly enriched in the presence of oil in combination with rhamnolipid or Slickgone. Furthermore, increased abundances of Colwellia and Oleispira, and stimulated bacterial production in microcosms with only rhamnolipid, Corexit, or Slickgone, indicated their involvement in biosurfactant/dispersant biodegradation. Our findings highlight varying microbial impacts resulting from rhamnolipid and chemical dispersants and suggest great promise for the application of biosurfactants in future marine oil spills.