Electrospinning is a well‐known technique for producing nanofibers using synthetic and natural polymers like mucilage. In this study, Plantago major Mucilage (PMM) was blended with polyvinyl alcohol (PVA) as a nontoxic adding agent, in order to produce electrospun nanofiber. Electrospinning parameters (voltage, tip‐to‐collector distance, feed rate, and PMM/PVA ratio) were optimized and solution properties were analyzed. The morphology of nanofibers was investigated using scanning electron microscopy (SEM), Fourier transform infrared (FTIR), X‐ray diffraction (XRD), and Brunauer–Emmett–Teller (BET). Mechanical strength of nanofibers was determined, and cell viability on nanofibers was discussed by MTT assay. The results of SEM indicated that the PMM/PVA (50/50) nanofibers obtained with average diameter of 250 nm. Viscosity, electrical conductivity, and surface tension of PMM/PVA solution were 550 Cp, 575 μS/cm, and 47.044 mN/m, respectively. FTIR and XRD results verified the exiting PMM in produced nanofibers and no chemical reaction between PMM and PVA. Improvement in mechanical strength and cell viability of nanofibers by adding PMM to PVA nanofibers indicated the potential application of PMM‐based nanofibers for medical and food industries. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47852.