Escalating demand for novel biocatalysts has intensified the production of vital enzymes from microbial, plant and animal origins. Microbial sources are mostly preferred owing to their short doubling time, capacity to utilize a wide range of substrates and easy maintenance. Enzymes have a crucial role in biorefiner ies mainly due to their ability to operate under mild conditions and substrate specificity, which enables efficient raw material biotransformation with low/no inhibitor formation, thus manifesting higher product yield. In this milieu, utili zation of recalcitrant lignocellulosic feedstocks may be considered as a sustain able alternative for fuel production. Enzymatic pretreatment and hydrolysis of lignocellulosics is being increasingly pursued globally for biofuel production with an objective to alleviate the disadvantages caused by physical, chemical and physicochemical approaches on the yield of the product, process economy and environment. Therefore, this chapter focuses on the industrially imperative micro bial enzymes and their applications in biorefineries for eco-friendly lignocellulosic fuel production. © 2018 John Wiley & Sons, Inc.