Abstract: In this work, a new, simple, cost-efficient and environmentally friendly water-based approach for the production of antibacterial nanocellulose/Cu₂O hybrid films was developed. The cuprous oxide nanoparticles were obtained by the microwave-assisted polyol synthesis using glycerol as a green reducing agent. The obtained samples were characterized using scanning electron microscopy (SEM) together with EDX analysis and elemental mapping, UV/Vis spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction analysis, dynamic light scattering. The results of UV/Vis spectroscopy confirmed the efficient synthesis of cuprous oxide nanoparticles showing a distinct absorption peak at 469 nm. The direct band gap energy of Cu₂O NPs is 2.10 eV. SEM images showed that Cu₂O NPs were well distributed in the nanocellulose matrix. Antibacterial activity of the samples was investigated against two gram-positive bacterial species, Bacillus cereus and B. thuringiensis, and gram-negative E. coli by a disk-diffusion and liquid broth tests. In the dark, the hybrid films showed good antibacterial activity against all tested bacterial strains. In liquid broth containing the hybrid films, B. thuringiensis was more sensitive than E. coli to Cu₂O NPs after 12 h of incubation. The Cu₂O NPs do not cause toxicity for mammalian cell cultures in the same concentrations, in which they are highly toxic for bacteria. The hybrid nanocellulose/Cu₂O films made of nontoxic, biodegradable reagents have a potential application in the field of treatment of wounds and skin infections. Graphic abstract: [Figure not available: see fulltext.].