Airborne transmission of infectious organisms is a considerable concern within the healthcare environment. A number of novel methods for ‘whole room’ decontamination, including antimicrobial 405 nm blue light, are being developed. To date, research has focused on its effects against surface-deposited contamination; however, it is important to also establish its efficacy against airborne bacteria. This study demonstrates evidence of the dose-response kinetics of airborne bacterial contamination when exposed to 405 nm light and compares bacterial susceptibility when exposed in three different media: air, liquid and surfaces. Bacterial aerosols of Staphylococcus epidermidis, generated using a 6-Jet Collison nebulizer, were introduced into an aerosol suspension chamber. Aerosolized bacteria were exposed to increasing doses of 405 nm light, and air samples were extracted from the chamber using a BioSampler liquid impinger, with viability analysed using pour-plate culture. Results have demonstrated successful aerosol inactivation, with a 99.1% reduction achieved with a 30 minute exposure to high irradiance (22 mWcm-2) 405 nm light (P=0.001). Comparison to liquid and surface exposures proved bacteria to be 3-4 times more susceptible to 405 nm light inactivation when in aerosol form. Overall, results have provided fundamental evidence of the susceptibility of bacterial aerosols to antimicrobial 405 nm light treatment, which offers benefits in terms of increased safety for human exposure, and eradication of microbes regardless of antibiotic resistance. Such benefits provide advantages for a number of applications including ‘whole room’ environmental decontamination, in which reducing levels of airborne bacteria should reduce the number of infections arising from airborne contamination.