The pursuit of sustainable energy has driven a shift from first-generation biofuels, derived from edible crops, to second-generation biofuels produced from non-food biomass such as forest residues and agricultural wastes which mitigates food-fuel competition and environmental concerns. However, relying on single feedstock leads to issues of seasonal availability and yield inconsistency. The concept of mixed lignocellulosic biomass (MLB) addresses these challenges by integrating diverse feedstocks, promoting resilient agricultural practices and enhanced soil health. Second-generation biofuel production involves key steps: pretreatment, saccharification and fermentation. Pretreatment, crucial for removing lignin and other components, enhances enzymatic hydrolysis efficiency. Advanced pretreatment methods, involving physical, chemical, physico-chemical and biological techniques, offer improved delignification and reduced inhibitory by-products. Combining methods such as steam explosion and biological pretreatment has shown promise in increasing fermentable sugar yields. This chapter explores the benefits and challenges of these methods, focusing on recent advancements and future prospects.