Abstract: Portulaca oleracea L. is a common plant which has received a lot of attention due to its superior antimicrobial properties. A nanofiber membrane loaded with Portulaca oleracea L. extract (POE) was prepared by electrospinning method using polyacrylonitrile (PAN) as raw material and N,N-dimethylformamide (DMF) as solvent. The nanofiber membrane morphology, chemical structure, mechanical properties, wettability, in vitro release properties, antioxidant and antimicrobial properties were characterized, and the antibacterial mechanism of PAN/POE nanofiber membranes was initially investigated. Results showed that the fiber diameter of the PAN/POE nanofiber membrane increased gradually with the increase of extract concentration, and the diameter was as high as (397.40±71.36) nm at POE mass fraction (w(POE)) of 12%. The mechanical properties of the nanofiber membrane increased with the increase of w(POE), and the fracture strength was up to (11.76±0.46) MPa. In addition, the cumulative release of POE in the nanofiber membrane increased with the increase of w(POE), up to (93.59±2.22)%. The scavenging rate of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was up to (87.08±4.43)%. The inhibition circle diameters of Staphylococcus aureus and Escherichia coli were up to (12.42±1.70) mm and (16.76±0.49) mm, respectively. POE is able to destroy the bacterial cell membranes and thus achieve the bactericidal effects through fluorescence staining experiments and SEM observations. The PAN/POE nanofiber membrane has great potential in the field of wound dressing.