In this study, rosemary essential oil was encapsulated in zeinelectrospun fibers at different concentrations of loading (0%, 2.5%, 5%, and 10% v/v). The chemical composition of rosemary essential oil was determined by GC-MS. The resultant zein-electrospun fibers were characterized by SEM, AFM, XRD, DSC, FTIR, and NMR. After being loaded with the essential oil, the fibers were evaluated for antimicrobial properties by the disc diffusion method against S. aureus (ATCC 1112) and E. coli (ATCC 1330). The release test was studied at pH values of 3 and 7.2 in phosphate buffer for 180 min. The GC-MS indicated that α-pinene occurred as a major compound in rosemary essential oil. Diameters of the zein-electrospun fibers increased in response to higher concentrations of rosemary essential oil. The AFM assay attributed a tubular morphology to the fibers. The physical status of rosemary essential oil in zein-electrospun fibers was determined by X-ray diffraction (XRD). DSC thermograms and FTIR spectra confirmed the existence of the rosemary essential oil in zein-electrospun fibers. FTIR spectra also indicated that adding rosemary essential oil to the fibers affected the secondary structure of zein protein. The NMR study showed that the electrospinning process did not change the secondary structure of zein. Disc diffusion indicated that zein-electrospun mats generated inhibition zones against S. aureus and E. coli. The release test revealed that pH values significantly affect the release of rosemary essential oil from fibers. The results demonstrated how loading zein-electrospun fibers with rosemary essential oil can benefit food packaging. Practical Application: In this study, electrospun fibers were produced from food-grade biopolymer to encapsulate rosemary essential oil. This product can be produced at industrial scale as an active food packaging/coating, controlled release, and delivery of the rosemary essential oil to food products and gastrointestinal. Also, it can be considered as a functional food to increase health.