Oil pollution in intertidal sediments is a serious environmental issue. The anoxic environment of the sediment hinders the decomposition of hydrophobic organic pollutants. Conventional bioremediation methods, such as the sole addition of electron acceptors, have struggled to achieve high efficiencies because of the low solubility of oil pollutants. Herein, the combination of anaerobically-synthesized rhamnolipids and electron acceptor was evaluated for the bioremediation of oil-polluted sediments. Meanwhile, the key genes involved in CNPS cycling were detected to understand the biogeochemical processes and the complex interactions between microbial metabolism, nutrient availability, and pollutant degradation. After the bioremediation, the combination of rhamnolipids and sulfate significantly enhanced the removal of polycyclic aromatic hydrocarbons (74.8 ± 1.4%). On day 270, the polyphenol oxidase activity of rhamnolipids and sulfate with rhamnolipids treatments reached 20870.1 ± 1988.7 mg/(kg·h) and 22373.8 ± 970.1 mg/(kg·h), respectively, which was significantly higher (P <
0.05) than other treatments. The sulfate consumption rate in the treatment with both sulfate and rhamnolipids consistently exceeded that of treatment with sulfate alone. Moreover, on day 60, the abundances of functional genes mediating sulfur oxidation (yedZ and soxY) were significantly higher in the combined treatment than in the sulfate group. The results revealed that the addition of rhamnolipids favored the growth of microorganisms and promoted S cycling, and the combination with sulfate dramatically enhanced the removal of polycyclic aromatic hydrocarbons. This study demonstrated that the combination of sulfate and rhamnolipids exhibited great potential on the anaerobic bioremediation of oil-contaminated intertidal zones.