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In this work, for the first time, zein nanofiber mats loaded with ethanol extracts propolis (EEP) were successfully produced. Raw propolis was extracted by ethanol 70% and total flavonoid content was estimated by aluminum chloride colorimetric method. The anti-microbial activity of the EEP was investigated and compared with amoxicillin via zone of inhibition test against various microorganisms included gram-positive: Staphylococcus aureus, Staphylococcus epidermidis, gram-negative: Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and fungus: Candida albicans. The EEP showed activity only against gram-positive types and fungus, whereas no activity was observed against gram-negative types. Electrospun zein nanofiber was obtained from 70% ethanolic solutions included different content of zein, 15–40 wt.%. The SEM images revealed a smooth ribbon-like morphology for zein …