Enzymatic hydrolysis of hemicellulose plays a crucial role in optimizing biofuel production, particularly in second-generation (2G) bioethanol processes. Xylanases, mannanases, and auxiliary enzymes, such as α-galactosidase, α-glucuronidase, acetylxylan esterase, ferulic acid esterase, and α-arabinofuranosidase, facilitate the breakdown of hemicellulosic structures, improving substrate accessibility for subsequent enzymatic actions. Recent studies have demonstrated the effectiveness of various microbial sources in bioethanol production, including Streptomyces sp., Pichia kudriavzevii, and Saccharomyces cerevisiae, which have been genetically modified or co-cultured to enhance fermentation efficiency. To make 2G bioethanol production processes economically effective, it is necessary to improve product yields and pursue cost-effective recovery and purification processes. Strategies such as enzyme rational design and the application of synergistic enzymatic systems have significantly improved sugar yield and ethanol production. Such advancements highlight the potential of enzyme engineering and microbial optimization in developing more efficient and sustainable 2G biofuel production processes. Therefore, this chapter will summarize the current state-of-the-art of hemicellulolytic enzymes, including aspects of their production, purification, optimization technologies, and current faced challenges, with emphasis on their application in 2G bioethanol production.