Due to the large specific surface area and rich pore structure, chitosan nanofiber membrane has many advantages over conventional gel-like or film-like products. However, the poor stability in acidic solutions and relatively weak antibacterial activity against Gram-negative bacteria severely restrict its use in many industries. Here, we present a chitosan-urushiol composite nanofiber membrane prepared by electrospinning. Chemical and morphology characterization revealed that the formation of chitosan-urushiol composite involved the Schiff base reaction between catechol and amine groups and the self-polymerization of urushiol. The unique crosslinked structure and multiple antibacterial mechanisms endowed the chitosan-urushiol membrane with outstanding acid resistance and antibacterial performance. After immersion in HCl solution at pH 1, the membrane maintained its intact appearance and satisfactory mechanical strength. In addition to its good antibacterial performance against Gram-positive Staphylococcus aureus (S. aureus), the chitosan-urushiol membrane exhibited synergistic antibacterial activity against Gram-negative Escherichia coli (E. coli) that far exceeded that of neat chitosan membrane and urushiol. Moreover, cytotoxicity and hemolysis assays revealed that the composite membrane had good biocompatibility similar to that of neat chitosan. In short, this work provides a convenient, safe, and environmentally friendly method to simultaneously enhance the acid resistance and broad-spectrum antibacterial activity of chitosan nanofiber membranes.