Bioremediation using microorganisms offers a sustainable approach to addressing hydrocarbon contamination. This study explores biosurfactant production by Yarrowia lipolytica IMUFRJ 50682 during crude oil and asphaltene-free fraction biodegradation in corn steep liquor (CSL)-based media. By evaluating CSL concentrations (5–30 g/L) and combinations with glucose, molasses, and crude oil, this study demonstrates that CSL is an effective nutrient source for supporting microbial growth and biosurfactant production. The highest emulsification index (EI = 73.3%) was achieved with 20 g/L of CSL after 48 h, while media containing mixed carbon sources (glucose and crude oil) enhanced metabolic efficiency, yielding a maximum cell growth of 8 g/L after 150 h. Despite inhibiting cell growth, the asphaltene-free fraction promoted biosurfactant activity, with the EI reaching 35.8% after 264 h. The results emphasize the importance of pH control, with the optimal emulsification being observed at pH ~6. This work highlights the potential of CSL as a cost-effective and sustainable additive, advancing applications in bioremediation and biosurfactant production and contributing to the development of environmentally compatible hydrocarbon degradation strategies.