A novel multifunctional biofilm was developed based on starch/whey protein isolate (WPI) combined with Ag2O nanoparticles (NPs). The biofilm was synthesized via the Lee-Meisel method, facilitating the production of silver nanoparticles. The resulting composite exhibits promising properties as an antioxidant, antibacterial, photocatalytic, and anticancer agent. The effect of different concentrations of Ag2O NPs was thoroughly investigated. The biofilm was comprehensively characterized, including thermal stability (TGA/DTA), light transmittance, film broadness, and water absorption capacity. X-ray diffraction (XRD) analysis and imaging studies were performed to assess the structural integrity and morphology of the biofilm. Dampness uptake and water vapor permeability studies were also conducted to evaluate the biofilm’s potential for real-world applications. Major weight loss of all three biofilms takes from 40 to 200 °C. The XRD pattern proves the presence of cubic and hexagonal phases of Ag2O NPs in biofilm. The 1 % Ag2O NPs showed higher cytotoxicity against A549 cells than 2 % Ag2O NPs with IC50 values of 15.66 ± 0.9 µg/mL and 32.14 ± 0.6 µg/mL, respectively. In Vero cells, 2 % Ag2O NPs showed the higher cytotoxicity with IC50 value 131.3 ± 0.3 µg/mL, followed by 1 % Ag2O NPs (IC50 = 185.6 ± 0.3 µg/mL). St/WPI/Ag2O 2 % shows better DPPH scavenging action, ranging from 70–73 %. The antibacterial activity of biofilm was investigated against S. aureus, E. coli, and C. albicans. The multi-functionality and biocompatibility of the film open new pathways for its use in biomedical, food packaging, bio-degradable plastic, and environmental fields.