Lignocellulosic biomass is an abundantly available, renewable, and inexpensive resource for biofuel production. The main constituents of lignocellulosic biomass are cellulose, hemicellulose, and lignin. Converting lignocellulosic biomass into biofuels requires pretreatment to separate these fractions. However, lignin, a complex polymer, hinders biomass conversion due to its recalcitrant nature and presents many challenges. Ligninolytic enzymes can transform the lignin component of lignocellulosic biomass. This chapter provides an overview of major ligninolytic enzymes, including laccases, lignin peroxidases, manganese peroxidases, and versatile peroxidases, which are effective in lignin degradation. Additionally, it highlights different sources for ligninolytic enzyme production and the mechanisms by which these enzymes break down lignin components. It also discusses the challenges and future perspectives of ligninolytic enzymes.