The synthesis of biodiesel via enzymatic means has drawn a lot of interest as a sustainable alternative to the conventional chemical-based techniques. Lipases are frequently used in the hydrolysis and transesterification processes, facilitating the effective synthesis of biodiesel from a variety of feedstocks, such as leftover cooking oil and inedible plant oils. However, phospholipids in crude oils have the ability to inhibit the lipase activity, thus lowering the quantity and quality of biodiesel. In order to overcome this challenge, phospholipases hydrolyze phospholipids, which improves the oil purity and thus lipase activity. In addition to enabling a more efficient biodiesel synthesis process, the combined action of lipases and phospholipases lessens the requirement for harsh chemical catalysts, which lowers energy consumption and environmental impact. This chapter provides an in-depth analysis of the enzymatic mechanisms, process optimization strategies, and recent advancements in integrating lipases and phospholipases for enhanced biofuel production. By highlighting their industrial applications and challenges, this work underscores the potential of enzymatic approaches in advancing sustainable bioenergy solutions.