The treatment and reuse of wastewater are crucial for the effective utilization and protection of global water resources. Polycyclic aromatic hydrocarbons (PAHs), as one of the most common organic pollutants in industrial wastewater, are difficult to remove due to their relatively low solubility and bioavailability in the water environment. However, biosurfactants with both hydrophilic and hydrophobic groups are effective in overcoming these difficulties. Therefore, a biosurfactant-producing strain Pseudomonas mosselii MP-6 was isolated in this study to enhance the bioavailability and biodegradation of PAHs, especially high-molecular-weight PAHs (HMW-PAHs). FTIR and LC-MS analysis showed that the MP-6 surfactant belongs to rhamnolipids, a type of biopolymer, which can reduce the water surface tension from 73.20 mN/m to 30.61 mN/m at a critical micelle concentration (CMC = 93.17 mg/L). The enhanced solubilization and biodegradation of PAHs, particularly HMW-PAHs (when MP-6 was introduced), were also demonstrated in experiments. Furthermore, comprehensive environmental stress tolerance tests were conducted to confirm the robustness of the MP-6 biosurfactant, which signifies the potential adaptability and applicability of this biosurfactant in diverse environmental remediation scenarios. The results of this study, therefore, have significant implications for future applications in the treatment of wastewater containing HMW-PAHs, such as coking wastewater.