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Abstract. Hemp (Cannabis sativa sp) is a medicinal plant that contains bioactive compound with antimicrobial properties, the incorporation of Hemp extracts into a nanofibrous film as an active food packaging solution has yet to be widely examined. In this study, Hemp extract was encapsulated into PVA electrospun fibers at different concentrations of loading (0%, 5%, 7%, and 9% v/v). The objectives of the present study were to fabricate an active nanofibrous film, evaluate its physiochemical properties and assess antibacterial effect on the selected foodborne pathogens. In this study, Hemp inflorescences grown at the Alabama A&M University, Winfred Thomas Agricultural Research Station, Huntsville, Alabama, were used. The whole samples were ground to particle sizes (250-850 µm). The ground Hemp material was macerated for 24 h at 25°C, centrifuged, filtered, and kept at 4°C until used for analysis. Polyvinyl alcohol solutions containing various concentrations of Hemp extract were electrospun with the Fluidnatek Electrospinner (LE-50, Nanoscience Instruments, Phoenix, AZ). The physicochemical properties of the electrospun nanofibers were characterized with SEM and FTIR, also viscoelastic properties of electrospinning solutions and properties of nanofibers which included nanofiber electrical conductivity, and mechanical properties were studied to assess nanofiber performance. After nanoencapsulation with extract in vitro and in situ antimicrobial activity were evaluated. Antibacterial activity against cocktails of enteric pathogens (Listeria monocytogenese (LM- H7969 serotype 4b, H7962 serotype 4b, and Scott A NADC 2045 Serotype 4b), and Salmonella enterica (SE- FSIS32105652-Typhimurium, FSIS32105654-Enteritidis, FSIS32105656- Infantis) was evaluated. All treatments were analyzed in triplicate and all statistical significance were test at 5%. FTIR results confirmed, within the nanofiber, the existence of cannabinoid, phenolic acid, and flavonoid chemical compounds. SEM images of Hemp loaded nanofibers showed uniform morphology, bead free, and smooth fibers, which ranged in diameter from 317±38.12nm (2a3) to 558.62±48.72nm (2a2). Electrical conductivity values ranged from 472±10.67µs/cm (2a1) to 714.76±27.41µs/cm (PVA neat); tensile strength values ranged from 3.11 MPa (2a1) to 7.74 MPa (2a2). Log 3.3 CFU/mL and log 3.0 CFU/mL reductions for Hemp extracts was observed against SE and LM, respectively. The number of SE and LM in the control groups reached 10.5 Log CFU/mL and 10.72 Log CFU/mL, respectively, at 37°C, while the number of SE and LM in Hemp nanofibers decreased to 9.5 Log CFU/mL and 8.87 Log CFU/mL after 24 h, respectively. In situ evaluation of nanofibers after four-day observation stored at 25°C showed that the Hemp nanofibers were bacteriostatic against SE. The results suggest that packaging raw poultry in Hemp nanofibers could help improve the raw storage shelf-life of poultry meat. Therefore, Hemp-loaded nanofiber will be a good candidate for food supply chain safety.