Abstract: This study compares the crude oil removal efficiency of free and biosurfactant-producing bacterial consortia (Bacillus tequilensis NR_104919 and Bacillus axarquiensis NR_115929) entrapped in chitosan (CH), chitosan/glutaraldehyde (CH/GA), and chitosan/Na-bentonite (CH/BNT) matrices. Additionally, the mechanical and thermal stability, swelling capacity, surface morphology, and chemical bonding of these composites were evaluated. Initially, the biosurfactant produced by the consortium was identified using screening methods, and its lipopeptide structure was confirmed through TLC and 1H NMR analyses, with a yield of 0.2 g/L. Following characterization, the biosurfactant-producing bacterial consortium was immobilized onto CH, CH/GA, and CH/BNT beads for further assessment. When comparing the oil removal capacities of the composites, the consortium immobilized in the CH/BNT matrix exhibited significantly higher crude oil removal efficiency than those entrapped in CH, CH/GA, and the free consortium. The maximum oil removal by the CH/BNT-entrapped consortium was achieved under optimized conditions: 0.5 mL crude oil, 0.1 g Na-bentonite, pH = 6.0, at 35 °C, over a 7-day incubation period. However, a decline in oil remediation performance was observed from the fourth reuse cycle onward. These laboratory-scale findings were further validated through ex-situ simulation and long-term experiments, confirming the potential of the CH/BNT composite for effective and sustainable petroleum pollutant remediation. The swelling capacity of inactivated CH was comparable to that of the CH/BNT composite, with both exhibiting greater swelling than CH/GA beads. Additionally, CH/BNT beads demonstrated superior mechanical stability relative to CH and CH/GA, which showed similar stability profiles. Thermal stability, surface morphology, and chemical bonding of the CH, CH/GA, and CH/BNT composites were characterized using TGA, FT-IR, and SEM analyses. CH/BNT composite represents an effective, economical, and environmentally benign alternative to costly synthetic materials for the remediation of oil pollution in aquatic environments. This study highlights the potential of chitosan/Na-bentonite-based composites as sustainable solutions for petroleum pollutant removal, while recommending pilot-scale investigations to assess their feasibility and performance under real-world conditions.