Novel physically crosslinked polyurethane (PUII), based on isophorone diisocyanates, was prepared by a conventional two-step method. The chemical structures of the PUII were characterized by fourier transform infrared (FTIR), proton nuclear magnetic resonance (1H NMR), gel permeation chromatography (GPC), scanning electron microscopy (SEM) and DSC. The PUII hydrogels were subjected to solvent-induced self-assembly in THF + water to construct a variety of morphologies. The self-assembly morphology of the PUII was observed by scanning electron microscopy (SEM). The PUII films with different amounts (0.2%, 0.4%, 0.6%, 0.8%, 1.0%) of 1,3,5-Tris(2-hydroxyethyl)hexahydro-1,3,5-triazine (TNO) were challenged with Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Gray mold. The results showed that when a small amount of antibacterial agent were added, the antibacterial effect of films on Botrytis cinerea was more obvious. The mechanical evaluation shows that the antimicrobial polyurethane films exhibit good mechanical properties.