The electrospinning technique was used for the nanofiber production of Alyssum lepidium mucilage with acetic acid and polyvinyl alcohol (PVA) polymer. Some parameters such as voltage, polymer concentration, tip-to-collector distance, and feed rate were optimised and applied for the fabrication of the nanofiber membranes of the seeds mucilage. The scanning electron microscopy images were used to find the optimised conditions for the electrospinning process. It was found that the aqueous solution of Alyssum mucilage/PVA (80:20), voltage (18kV), polymer concentration (50%), tip-to-collector distance (10cm) and feed rate (0.125ml/h) could be successfully used to obtain uniform nanofibers with diameters as low as 139.9nm. X-ray diffraction and Fourier transform infrared spectrometer analysis also proved the presence of the alyssum mucilage/PVA nanofiber. In this study, the used electrospun procedure was biodegradable, inexpensive, non-toxic, and maintainable enough to optimise the mucilage nanofiber fabrication as a new source, thereby improving the potential application of the nanofiber biomembrane in filtration and medical systems with biocompatible and biodegradable properties.