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Biodegradable nanofilms from polyvinyl pyrrolidone (PVP), carboxymethyl cellulose (CMC), citric acid (CA), glycerol (G), and zinc oxide nanoparticles (ZnO-NPs) were prepared using different ZnO concentrations and different electron beam irradiation doses, enabling crosslinking formation. The prepared films were characterized by X-ray diffractometer, Fourier transform infrared spectroscopy, thermogravimetric analyser, and transmission electron microscopy. The swelling percentage of PVP:CMC films was ordered in the sequence of composition ratio 1:2 > 1:1 > 2:1. Results showed decrease in swelling capacity accompanied by increase in gelation percentage of (PVP:CMC)/CA/G)/ZnO nanofilms as the irradiation dose increased up to 20 kGy. The tensile strength of (PVP:CMC) films increased by the incorporation of ZnO-NPs and increasing the irradiation dose. The thermal stability of the prepared (PVP:CMC)/CA/G/ZnO nanofilms was enhanced as the irradiation dosage increased. The water vapour transmission rate of the irradiated films was decreased. The biodegradability of the prepared nanofilms was monitored during 16 weeks and it exceeded 65% weight loss from the original blank weight. Moreover, the nanofilms exhibit antimicrobial activity against fungi, Gram-negative, and Gram-positive bacteria. The broad antimicrobial activity spectrum of the prepared nanofilms increased as the concentration of ZnO-NPs increased. These results suggested that (PVP:CMC)/CA/G/ZnO nanofilms can serve as biodegradable materials in various applications characterized by antimicrobial activity.