In this study, Ajowan essential oil was encapsulated in electrospun zein fibers at different amounts of loading (0, 22, 44, and 88 mg/ml). The resulting electrospun zein fibers containing Ajowan essential oil were characterized by scanning electron microscope (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD), differential Scanning Calorimetry (DSC), fourier transform infrared (FTIR). Antimicrobial properties of fibers against Staphylococcus aureus and Escherichia coli were also determined by the disc diffusion method. GC/MS analysis was employed to identify the constituents of the Ajowan essential oil. The diameters of electrospun zein fibers increased with increasing concentration of Ajowan essential oil. Physical status of Ajowan essential oil in electrospun zein fibers was determined by XRD. Thermal properties of loaded electrospun fibers were studied by DSC. FTIR approved the encapsulation of essential oil in electrospun zein fibers. The encapsulation efficiency of Ajowan essential oil in electrospun zein fibers was about 100%. Antimicrobial results showed that the Ajowan essential oil loaded electrospun fibers were effective against S. aureus and E. coli. This could be due to the major constituents of Ajowan essential oil. Ajowan essential oil was successfully encapsulated in electrospun fibers for the first time.