To overcome the barriers often met by traditional ophthalmic formulations, polymeric films can be utilized as an alternative to enhance drug retention duration while managing medication release. In the current investigation, polymeric films made of poly (vinyl) alcohol (PVA) and chitosan (CS) loaded with Moxifloxacin Hydrochloride (M-HCl) and plasticized with Glutaraldehyde were formulated as potential ophthalmic delivery for the treatment of conjunctivitis. The thickness, surface pH, opacity, folding endurance, and % hemolysis were measured, followed by the transparency, microscopy, electrical conductivity, mechanical strength, swelling index, and invitro drug release studies. FTIR spectroscopy further accessed the interactions between the polymers and drug molecules. The thermal behaviour and diffraction pattern of the films were evaluated using DSC and XRD studies. Lastly, the antimicrobial effectiveness of the M-HCl-loaded films was studied against P. aeruginosa and S. aureus. The rabbit eye irritation study conducted in vivo confirmed that the film was comfortable for use in ocular applications. Upon integrating the findings, it was determined that the optimal film formulation was PC3 (PVA: CS = 7:3), exhibiting superior transparency, heightened intermolecular hydrogen bonding, elevated mechanical strength, increased crystallinity, larger crystal size, optimal swelling index, a high percentage of controlled drug release (%CPDR), and the highest antimicrobial activity.