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 againstEscherichia coli(E. coli) andStaphylococcus aureus(S. aureus) was investigated, and the samples loaded with ɛ-PL demonstrated stronger antimicrobial activity againstS. aureus.