In this study, a series of collagen-chitosan-eugenol (CO-CS-Eu) flow-casting composite films were prepared using collagen from sturgeon skin, chitosan, and eugenol. The physicochemical properties, mechanical properties, microstructure, as well as antioxidant and antimicrobial activities of the composite membranes were investigated by various characterization techniques. The findings revealed that the inclusion of eugenol augmented the thickness of the film, darkened its color, reduced the transparency, and enhanced the ultraviolet light-blocking capabilities, with the physicochemical properties of the CO-CS-0.25%Eu film being notably favorable. Eugenol generates increasingly intricate matrices that disperse within the system, thereby modifying the optical properties of the material. Furthermore, the tensile strength of the film decreased from 70.97 to 20.32MPa, indicating that eugenol enhances the fluidity and ductility of the film. Added eugenol also exhibited structural impact by loosening the film cross-section and decreasing its density. The Fourier transform infrared spectroscopy results revealed the occurrence of several intermolecular interactions among collagen, chitosan, and eugenol. Moreover, the incorporation of eugenol bolstered the antioxidant and antimicrobial capabilities of the composite film. This is primarily attributed to the abundant phenolic/hydroxyl groups present in eugenol, which can react with free radicals by forming phenoxy groups and neutralizing hydroxyl groups. Consequently, inclusion of eugenol substantially enhances the freshness retention performance of the composite film. PRACTICAL APPLICATION: ● The CO-CS-Eu film utilizes collagen from sturgeon skin, improving the use of sturgeon resources.● Different concentrations of eugenol altered its synergistic effect with chitosan.● The CO-CS-Eu film is composed of natural products with safe and edible properties.