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In recent years, the synthesis of nanofibers using plant extracts and bioactive materials has been extensively studied and recognized as a suitable and efficient method applicable in the food packaging field. In this research, an antimicrobial material was introduced by the immobilization of glucose oxidase (GOx) in Nylon–Ag masterbatch/chitosan/Rosmarinus officinalis extract nanofiber via electrospinning technology. Nylon–Ag masterbatch/chitosan/Rosmarinus officinalis composite nanofibrous membranes with an average diameter of 207 ± 18 nm were successfully prepared using the electrospinning technique. The chemical properties of membranes were analyzed by Fourier transform infrared spectroscopy (FTIR) and the morphological characterization of nanofibers was evaluated with field emission scanning electron microscopy (FE-SEM). Moreover, enzymatic activity of GOx was determined by the Carmine method. FTIR results showed the successful incorporation of glucose oxidase and Rosmarinus officinalis into the nanofiber composite. Immobilized GOx showed high (79.5%) enzymatic activity in the optimum sample. The Rosmarinus officinalis, glucose oxidase-incorporated Nylon–Ag masterbatch/chitosan nanofibrous exhibited excellent antimicrobial activity on both gram-negative bacterium Escherichia coli (97.5%) and gram-positive bacterium Staphylococcus aureus (99.5%). The antibacterial and antioxidant Nylon–Ag masterbatch/chitosan/Rosmarinus officinalis/GOx nanofibrous membrane showed higher potential, compared to the control sample, to be used as food packaging by improving the shelf life and maintaining the quality of food stuffs. Therefore, this research recommends it as a promising candidate for food preservation applications. [ABSTRACT FROM AUTHOR]