Lignocellulosic biomass presents a renewable resource for biofuel production; however, its recalcitrant nature poses a significant challenge to its sustainable use. This chapter explores the critical role accessory enzymes could play in overcoming the challenges of breaking down the rigid, complex structure of lignocellulose, which is predominantly composed of cellulose, hemicellulose, and lignin, to fermentable sugars. In contrast to primary enzymes that directly catalyze the hydrolysis of the substrate, accessory enzymes enhance efficiency by degrading the complex structures of the biomass, thus increasing substrate availability while mitigating inhibitory effects. Enzymes such as β-glucosidases, hemicellulases, pectinases, and lytic polysaccharide monooxygenases work synergistically with primary enzymes to improve biomass hydrolysis. Furthermore, these enzymes reduce the reliance on harsh chemical pretreatments and pave the way for greener, cost-effective biomass conversion. The chapter also discusses lignocellulolytic enzyme classification, their microbial sources, industrial applications, and emerging omics-based discovery methods like metagenomics, proteomics, etc., highlighting their potential to revolutionize the search for better accessory enzymes, which could eventually be utilized to convert biomass into biofuels.