Nanotechnology has transformed numerous industrial processes, including the generation of renewable energy sources like biofuels. The complex and recalcitrant structure of the raw materials presents significant challenges for biofuel production, particularly from lignocellulosic biomass. To enhance the availability of fermentable sugars, lignocellulosic biomass—which is mostly composed of cellulose, hemicellulose, and lignin—needs to be well pretreated. Because traditional pretreatment techniques are frequently expensive, energy-intensive, and environmentally harmful, new effective and sustainable treatments must be developed. From biomass pretreatment to conversion into useful fuels, nanotechnology offers creative ways to improve the entire biofuel production process. This chapter explores the vital role that nanomaterials play in enhancing the production of biofuel, with an emphasis on lignocellulosic biomass pretreatment. Because of their considerable benefits in catalysis and enzyme immobilization, nanomaterials like metallic nanoparticles, carbon-based nanostructures (such as carbon nanotubes (CNTs) and graphene), and magnetic nanoparticles (MNPs) are suitable for biofuel applications. Metallic nanoparticles, such as those derived from cobalt (Co), nickel (Ni), and platinum (Pt), function as effective nanocatalysts in procedures like transesterification, which is crucial for the manufacture of biodiesel. Additionally, the use of magnetic nanoparticles makes it simple to collect and reuse enzymes, which greatly reduces operating costs and improves the sustainability of the process. With a focus on the use of nanomaterials in lignocellulosic biomass pretreatment, this chapter explores the latest developments in nanotechnology applications in the manufacture of biofuel. The role of nanocatalysts, nanoenzymes, and nanostructured materials is investigated in depth, with a focus on enhancing the efficiency, sustainability, and scalability of biofuel production processes. Nanotechnology has enormous potential for the future of biofuel production by improving process efficiency and lowering environmental impact, supporting global efforts to move toward more sustainable energy sources.