The involved components of formulated blending, CNF and CuO NPs, were individually obtained by using a biorefinery strategy for agricultural waste valorization together with an optimized chemical precipitation, assisted by ultrasounds. The optimization of synthesis parameters for CuO NPs have avoided the presence of undesirable species which usually requires of later thermal treatment with associated costs. The aerogels-based structure, obtained by conventional freeze-drying, acted as 3D support for CuO NPs providing a good dispersion within the cross-linked structure of the nanocellulose and facilitating direct contact of the antibacterial phase against undesirable microorganisms. All samples showed a positive response against Escherichia coli and Staphylococcus aureus. An increase of the antibacterial response of the aerogels, measured by agar disk diffusion test, has been observed with the increase of CuO NPs incorporated, obtaining width of the antimicrobial “halo” (nwhalo) from 0 to 0.6 and 0.35 for S. aureus and E. coli, respectively. Furthermore, the aerogels have been able to deactivate S. aureus and E. coli in less than 5 hours when the antibacterial assays have been analyzed by broth dilution method. From CNF-50CuO samples, an overlap nanoparticles effect produced a decrease of the antimicrobial kinetic.