This chapter mainly focuses on different non-food-based plant lignocellulosic biomass for biofuel production which are otherwise considered waste. This is an alarming call of the present scenario to overcome the high fuel cost and environmental pollution. Biofuel consumption is very high compared to its production. Many technological gaps are there that increase production costs and reduce the yield. Here, a biological pre-treatment process is discussed that is more environment friendly, and its end product can be employed straightaway for ethanol production via fermentation without generating any harmful residues. The diversity of microbes has been reported to produce lignocellulosic degrading enzymes. This low-efficient process can become cost-efficient by genetically modifying these microorganisms for high yield, quality improvement, and less time consumption for industrial application.