The biofuel industry has made significant progress in replacing fossil fuel and has reached the stage of commercial application. However, biofuel still falls far short of fully replacing fossil fuels, and this can be attributed to economic constraints, environmental concerns, and technological limitations. To deal with this, researchers have done extensive studies on the minute details of improving the efficiency of upstream and downstream processes of biofuel production. The application of nanotechnology-based processes and products has shown remarkable improvement in biofuel production. The use of nanoparticles in the form of catalyst during upstream and downstream processes was found to improve production, product yield, and quality of biofuels. Newer approaches are being made to deal with the containment points in the biofuel industry by applying nanotechnology. This chapter specifically discusses the upstream and downstream processes of biofuel production that involve catalysts/enzymes and elaborates on the role of nanotechnology in improving the overall output as compared to conventional modalities.