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Background: Composite nanofiber films loaded with ε-polylysine (PL) and gallic acid (GA) were prepared using a zein/gelatin (ZG) electrospinning method to develop effective active packaging films for tuna preservation. The morphology, structure, thermal stability, hydrophobicity, antibacterial, and antioxidant properties of the films, and their application for tuna during a period of storage of 4 °C were investigated., Results: PL reduced the average diameter of ZG fibers, whereas GA increased it. The PL/GA/ZG film possessed a well distributed fiber morphology with an average diameter of 810 ± 150 nm. Fourier-transform infrared spectroscopy and X-ray diffraction results showed the physical loading of PL and GA in ZG film with the main chemical bonds and crystal structure unchanged. The addition of both PL and GA reduced hydrophobicity of the ZG film while the PL/GA/ZG film was still hydrophobic. GA enhanced its thermal stability and contributed to its antioxidant activity. PL and GA synergetically enhanced the antibacterial activity of ZG film against Shewanella putrefaciens. PL combined with GA is more suitable for modifying ZG film than GA alone. The PL/GA/ZG film effectively inhibited total viable counts, total volatile base nitrogen, fat oxidation, and texture deterioration of tuna fillets at 4 °C storage, and could extend the shelf life by 3 days., Conclusions: The PL/GA/ZG nanofiber film demonstrated promising potential for application in the preservation of aquatic products as a new antibacterial and antioxidant food packaging. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)