Considering the increasing resistance of numerous bacteria to antibiotics, a novel wound dressing material was developed with naturally acquired olive leaf extract, which shows not only good antimicrobial activity, but also very good antioxidant activity. Besides that, the leaves are treated as waste in agriculture, giving an impact on waste management. An environmentally friendly procedure, electrospinning, was used for the first time to prepare polysaccharide nanofibrous mats with incorporated olive leaf extract, with the unique property of releasing the active phenolic components in a prolonged manner over 24 hours. The developed electrospun mats were characterized using scanning electron microscopy, high-performance liquid chromatography and ultraviolet-visible spectroscopy for determination of free radical scavenging activity by 2,2-diphenyl-1-picrylhydrazyl, antimicrobial testing and release kinetics. Antimicrobial tests have shown that electrospun mats with olive leaf extract achieve reduction towards the tested microorganisms: Staphylococcus aureus (G+), Escherichia coli (G-), Enterococcus faecalis (G+) and Pseudomonas aeruginosa (G-), while the high antioxidant activity of olive leaf extract was preserved during the electrospinning procedure. Release of olive leaf extract from electrospun mats was mathematically modeled, and the release kinetics evaluation indicates the appropriateness of the Korsmeyer-Peppas model for fitting the obtained results of release ability due to erosion of polysaccharide nanofiber mats.