In this study, biopolymer of chitosan-based films were synthesized, which were impregnated with zinc oxide nanoparticles (ZnO NPs) at concentrations of 0, 1, 5 and 10 % w:v to obtain a film with microbicide properties and non-toxic for humans. The ZnO NPs were phytosynthesized with ethanolic extract of Ruta graveolens, by UV-Vis spectrophotometry and Tauc equation were estimated their Band gap energy = 3.37 eV at wavelength of 302 nm. The films characterization by FTIR spectroscopy, swelling test, TGA and DSC indicated that the linking of ZnO NPs with the biopolymer was successfully developed. Finally, to evaluate the potential application of the biopolymer in biomedicine, the microbicidal activity against pathogens Escherichia coli, Staphylococcus aureus and Candida albicans was evaluated. The analyses revealed that the microorganisms are susceptible to the doses of ZnO NPs present in the biopolymer. Resulting the biopolymers with 5 % w:v of ZnO NPs being the most efficient for inhibition zones of 33, 31 and 27 mm that were obtained for each microorganism respectively.