The preparation of poly (vinyl alcohol)/chitosan/ZnO (PVA/Cs/ZnO) nanocomposite films as bioactive nanocomposites was implemented through an environmentally friendly approach that included mixing, solution pouring, and solvent evaporation. The nanocomposite films were characterized using various techniques such as X-ray diffraction (XRD), Fourier-transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and UV-Vis spectroscopy. The XRD study revealed the encapsulation of nanoparticles by the PVA/Cs blend matrix. The DSC results showed that the addition of ZnO NPs increased glass transition and melting temperature values of the PVA/Cs blend. ATR-FTIR spectra detected an irregular shift (either red or blue) in some of the characteristic bands of the PVA/Cs nanocomposite, indicating the existence of intra/intermolecular hydrogen bonding creating an interaction between the OH groups of PVA/Cs and ZnO nanoparticles. A thermogravimetric (TGA) analysis demonstrated that the nanocomposites achieved better thermal resistance than a pure PVA/Cs blend and its thermal stability was enhanced with increasing concentration of ZnO nanoparticles. UV analysis showed that with an increase in the content of ZnO NPs, the optical bandgap of PVA/Cs was decreased from 4.43 eV to 3.55 eV and linear and nonlinear parameters were enhanced. Our optical results suggest the use of PVA/Cs/ZnO nanocomposite films for various optoelectronics applications. PVA/Cs/ZnO nanocomposites exhibited significant antibacterial activity against Gram-positive and Gram-negative bacteria. It was found that nanocomposite samples were more effective against Gram-positive compared to Gram-negative bacteria.