Cellulose is lack of UV-blocking and antibacterial properties, which have limited its application. In this work, the nanoscale lignin with high content of hydroxyl groups and small particle size in prehydrolysate was isolated and used as a green reinforcement ingredient for fabrication of cellulose nanofibril (CNF) films with excellent mechanical properties, as well as UV protection and antibacterial capabilities. Cryogenic transmission electron microscopy (Cryo-TEM) and nuclear magnetic resonance analyses showed that the resulting lignin was in the form of nanoparticles (6–12 nm) with high phenolic hydroxyl contents (4.9 mmol/g). The optimum lignin inclusion rate of 5% allowed it to reinforce CNF composite film, increasing its tensile strength from 108.5 to 143.3 MPa. In addition, the film exhibited excellent UV protection capabilities. It blocked 91.5% of UV-A and 99.9% of UV-B light. Finally, the resulting lignin-based CNF films exhibited antibacterial activities against both Escherichia coli and Streptococcus hemolyticus . This work demonstrates the utility of nanoscale lignin from prehydrolysate can be used to produce cellulose-based composite films with valuable properties.