Biofuels are broadly defined as fuel products derived from biomass, including butanol, ethanol, biogas, H2, and biodiesel. In simple terms, bioalcohols and biodiesel are used mainly as petroleum additives or replacements, whereas biogases are used mainly for cooking and heating (Taylor et al., 2012). Biomass used as raw material for biodiesel can refer to any organic substrate, such as sugarcane, corn, wheat, organic, agricultural or municipal waste, algae, lignocellulosic wastes, etc. (Taylor et al., 2012). Edible crops are mainly used for production of first-generation biofuels and some are already commercially available (Havlík et al., 2011). The yield of bioethanol, produced from sugarcane in Brazil and corn in the United States, increased from 31.3 billion liters in 2005 to over 85.6 billion liters in 2010 (Carriquiry et al., 2011). However, concerns around the suitability of these biofuels led to the development of second-generation biofuels, produced from non-food biomass that has emerged as a promising and more energy-efficient and flexible alternative to first-generation biofuels (Havlík et al., 2011). Traditionally, mesophilic organisms and their enzymes have been chosen for the production of biofuels, although in recent years extremophiles have emerged as promising alternatives, due to their robustness and versatility. Versatility can be innate or engineered, thanks to the increasing knowledge of metabolic pathways and the growing number of genetic tools available for these organisms.This chapter gives an overview of the role that extremophiles and their enzymes play in biofuel production, with special emphasis on thermophilic organisms. The various processes (including pretreatment, cellulose and hemicellulose hydrolysis as well as consolidated bioprocessing), used for biofuel production, as well as the status of different biofuels (bioethanol, biobutanol, biogas and biodiesel) is reviewed. Major microbial organisms/strains and enzymes involved in the different steps during biofuel production are mentioned and new developments in strain engineering, enzyme modification and feedstock treatment are discussed.