The battle against the prevalence of hospital-acquired infections has underscored the importance of identifying and maintaining the cleanliness of possible infection transmission sources in the patient's environment. One of the most crucial lines of defense for mitigating the spread of pathogens in a healthcare facility is the removal of microorganisms from the environment by air filtration systems. After removing the pathogenic microorganisms, the filters used in these systems can serve as reservoirs for the pathogens and pose a risk for secondary infection. This threat, combined with the ever-growing prevalence of drug-resistant bacterial strains, substantiates the need for an effective bactericidal air filter. To this end, a broad-spectrum bactericidal polyurethane incorporating immobilized quaternary ammonium groups was developed for use as an air filter coating. In this study, the bactericidal activity of the polymer coating on high-efficiency particulate air (HEPA) filter samples was quantified against eight bacterial strains commonly responsible for nosocomial infection-including drug-resistant strains, and confirmed when applied as a filter coating in conditions mimicking those of its intended application. The coated HEPA filter samples exhibited high bactericidal activity against all eight strains, and the polyurethane was concluded to be an effective coating in rendering HEPA filters bactericidal.