Environmentally friendly waterborne polyurethane (WPU) composites were developed from polycarbonate diol (PCDL) and isophorone diisocyanate (IPDI), using 3-dimethylamino-1,2-propanediol (DMAPD) as a chain extender, followed by quaternizing by 1-bromodecane and 1-bromododecane, respectively. The quaternary ammonium salt WPU emulsions were obtained by the addition of appropriate amounts of water under high-speed stirring, which had a small particle size and better dispersion stability. Because of the antibacterial characteristics of the quaternary ammonium compound, these WPU films showed high efficiency in killing Gram-negative Escherichia coli and Gram-positive Staphylococcus epidermidis. The WPU with a longer hydrocarbon tail offered higher antimicrobial potency because of strong hydrophobic interactions. The antimicrobial action of the WPU films was based on contact killing, without leaching of bactericidal species, as revealed by a zone-of-inhibition test. Moreover, the WPU films exhibited high mechanical properties with a tensile strength of 36 MPa and an elongation at break of 620%. Therefore, these WPU composites hold great promise for antibacterial applications in coatings.