Biofuels are produced from renewable sources, including crops, agricultural subproducts, and animal waste. They are considered essential for transitioning to a low-carbon economy due to their capacity to reduce greenhouse gas emissions. In contrast to fossil fuels, biofuels are regarded as carbon-neutral because the carbon dioxide generated by combustion is absorbed by plants, making them a promising solution for both transportation and energy production. In addition, biofuels contribute to greater energy security by reducing reliance on imported fossil fuels. Second-generation bioethanol, which is produced from renewable sources, passes through biomass pretreatment and enzymatic hydrolysis for further fermentation. Biodiesel production involves oil transesterification through chemical or enzymatic catalysis. In both cases, the enzymatic choice benefits from their specificity and mild conditions, which require less energy. Biofuel enzyme market is continuously growing, and new technologies are being developed to enhance their efficiency and applicability. Supporting domestic biofuel production allows countries to enhance their energy independence and minimize the risks tied to the instability of global oil markets. This chapter provides an overview of the role of enzymes employed in the biofuel industry to enhance the efficiency and sustainability of biofuel processes.