<p>Biodegradable packaging offers a sustainable approach to extend shelf-life of food and reduce environmental impact. In this study, a bilayer active film composed of Persian acorn starch and zein nanofibers incorporating 10% (v/w) cinnamon essential oil (CEO) was developed via casting and electrospinning methods. Cinnamaldehyde (83.81%) was the dominant constituent of CEO, which exhibited the strongest antibacterial activity, particularly against <i>Staphylococcus aureus</i> with minimum inhibitory concentration of 0.25 µL/mL. The physicochemical properties of the extracted acorn starch and the films were investigated. The scanning electron microscopy (SEM) of zein nanofiber showed that the mean fiber diameter decreased from 521 to 497 nm with the addition of CEO. While added CEO in zein nanofiber decreased the tensile strength (35.36%), Young's modulus (19.44%) and water vapor permeability (28.38%), but significantly increased elongation at break (37.94%), hydrophobicity, and antioxidant properties of the film. Fourier transform infrared spectroscopy (FTIR) confirmed hydrogen bond formation between layers and effective CEO encapsulation within the zein nanofiber matrix. CEO release was medium-dependent, with the slowest rate was observed in distilled water. Acorn starch/zein/CEO film significantly reduced lipid oxidation (by 44%), inhibited total mesophilic bacteria, and extended the shelf-life of rainbow trout fillets by 6 days. Overall, the active bilayer starch/zein film demonstrates promising potential as a bio-based active packaging material for fish fillet preservation.</p> Graphical Abstract <p></p>