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A new natural and environmental friendly wound dressing was introduced for the first time that was prepared by electrospinning method. This new wound dressing has chitosan base, and poly (ethylene oxide) was added as co-spinning agent to improve spinnability of chitosan. Moreover, thyme extract as a natural antibacterial additive was introduced in the as electrospun nanofibers scaffold in order to increase those wound healing properties. Some parameters of electrospinning such as feed rate, nozzle-collector distance, voltage and content of thyme extract in nanofiber structure were studied and optimized. The average diameters of prepared nanofibers was determined by “Clemex vision professional edition” software. Morphology and structure of electrospun nanofibers was studied with use of scaning electorn microscopy and Fourier transform infrared spectroscopy spectroscopy. The results showed that the antibacterial activity of nanofibers increased as the amount of thyme extract was increased, thus a chitosan/PEO containing 3% of thyme extract was selected as the best prepared nanofiber for wound dressing preparation. Chitosan/PEO/thyme nanofiber showed high stability in the buffer and good antibacterial activity against three understudy bacteria including Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus.