In the present study, electrospun nanofiber mats were fabricated by mixing different ratios (96:4, 95:5, 94:6, 93:7, and 92:8) of Persian gum (PG) and poly (ethylene oxide) (PEO). The SEM micrographs revealed that the nanofibers obtained from 93% PG and 7% PEO were bead-free and uniform. Therefore, it was selected as the optimized ratio of PG:PEO for the development of antimicrobial nanofibers loaded with ɛ-Polylysine (ɛ-PL). All of the spinning solutions showed pseudoplastic behavior and the viscosity decreased by increasing the shear rate. Additionally, the apparent viscosity, G', and G″ of the spinning solutions increased as a function of PEO concentration, and the incorporation of ɛ-PL did not affect these parameters. The electrical conductivity of the solutions decreased when increasing the PEO ratio and with the incorporation of ɛ-PL. The X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) spectra showed the compatibility of polymers. The antimicrobial activity of nanofibers against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was investigated, and the samples loaded with ɛ-PL demonstrated stronger antimicrobial activity against S. aureus.