Xylanases are hydrolytic enzymes secreted by different strains of bacteria and fungi. They are responsible for the hydrolysis of xylan into monomeric xylose. Xylanases have wide industrial applications in baking industry, wood industry and laundry detergents production. One of the most important applications of xylanases is second-generation biofuel production where they serve as hydrolytic enzymes for lignocellulosic biomass in the saccharification step to produce sugars for the subsequent fermentation. The second generation biofuel production especially biohydrogen, bioethanol and biobutanol using xylanases received great attention in the last two decades. This chapter spotlights the efforts exerted in the last two decades for the biofuels production using xylanase enzymes.