Biodegradable materials have overtaken the nanofiber research field due to their sustainability and promising properties while upscaling is still a challenge. Equipment suitable for mass production with a fixed wire electrode was used for the electrospinning of fiber membranes. This research reports the preparation of novel nanofibers membranes with high propolis content. Electrospinning solutions with combinations of different propolis extracts and an adapted mixing method were used to obtain fibers. The morphology was examined by scanning electron microscopy and various inclusions have been detected, propolis incorporation was analyzed by optical microscopy and depend on propolis extracts used, inclusions were found in an amorphous state. The propolis content was calculated and reaches 83%, membranes have the characteristic color and smell of propolis. The interaction of components was studied by Fourier-transform infrared spectroscopy. Since propolis fiber samples showed an antiviral effect against the SARS-CoV-2 virus (about 2 log decrease in viral titer compared to the reference) and an antibacterial effect against Staphylococcus aureus and Salmonella enterica bacteria (bacteria were not observed both under and around the samples), obtained fibrous membranes have foreseeable potential applications of bioprotection and may provide an instructive microenvironment for medical treatment, bioactive delivery systems, filter material and wound protective layers.