Abstract: To explore the feasibility of polysuccinimide (PSI) electrospun fibrous membranes as antimicrobial preservative films, PSI/polyurethane (PU) fibrous composite membranes were prepared via co-electrospinning with blended and layered structure to enhance their mechanical properties. And crosslinking modification of the PSI component was performed to further improve the mechanical strength and impart antimicrobial functionality. The effects of the fabrication methods and crosslinking on membranes’ micromorphology, chemical structure and mechanical properties were investigated. Furthermore, the hydrophilicity, air permeability, water vapor permeability, antimicrobial activity, cytotoxicity, and strawberry preservation performance of the modified membranes were evaluated. The results indicate that the introduction of PU fibers can effectively improve the strength of membranes. The crosslinking treatment opens the succinimide rings in PSI to form crosslinking structure and cationic amino groups, which significantly enhances the mechanical properties of membranes and endows them with antibacterial activity. All the modified membranes exhibit a 100% inhibition rate against Escherichia coli and over 95% against Staphylococcus aureus. Moreover, they demonstrate favorable hydrophilicity, water vapor permeability, and cytocompatibility (cell viability >90%). Notably, the modified PSI/PU blended membranes exhibit optimal mechanical properties and air permeability, and the tensile strength increase from 0.35 ± 0.05 MPa to 1.67 ± 0.2 MPa after modification. In strawberry preservation experiments, the PSI/PU blended membranes effectively delayed spoilage due to their balanced air permeability, good water vapor permeability, and potent antimicrobial properties, demonstrating significant potential for applications in food packaging.