By covalently immobilizing imidazolium ion onto molecular chain, functional polyurethane (PU) is fabricated and thus an effective way is initiated to prepare electrospun membranes with antibacterial activity. In the experiment, PUs containing imidazolium ion side group (Bmim-PUs) are synthesized through a two-step polymerization process. It includes prepolymerization of isophorone diisocyanate (IPDI) with polyester glycol and chain extension polymerization using imidazolium-based ionic diol (Bmim-OH). Then, the obtained Bmim-PUs are electrospun into fibrous membranes with a diameter of similar to 640 nm. After a careful assessment, antibacterial activities of electrospun membranes against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli are clearly exhibited. The antibacterial efficiency of Bmim-PUs on both bacteria species improves by 60% in comparison with PU without imidazolium ion. This research suggests a simple but effective methodology to design and fabricate ultrafine fibrous membrane with significant antibacterial activity. Moreover, the obtained fibrous membranes have widely potential applications in protective textiles, filtration, and biomedical engineering.