Abstract Introduction/Objective Hand hygiene (HH) decreases healthcare-associated infections (HAI). Available products include alcohol-based gels, foams, wipes, and “gold-standard” hand-washing with soap and water. We tested an investigational device (HyLuxO3; GMI, LLC, patent pending) for antimicrobial effect (AME). HyLuxO3 was engineered to deliver UV-C light energy and high velocity O3 airflow to safely achieve human skin antisepsis within OSHA and EPA regulatory limits. Combined UV and O3 has yet to be evaluated for HH and may demonstrate synergistic AME. Methods HyLuxO3 was tested on LB agar to titrate device variables to ascertain intensities for optimal AME; later testing was performed on VITRO-SKIN (Florida Suncare Testing, Bunnell, FL), a human skin surrogate. ATCC strains of MRSA, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans were used to test AME vs. vegetative microbes; Bacillus atrophaeus spores were used as a surrogate for C. difficile. Tested variables included time under device, [O3], airflow velocity, 222 and/or 254 nm UV light, sample distance from UV lamp, and UV beam width. Positive controls were used to calculate log-kill curves for AME. Results Similar results were seen on LB agar and VITRO-SKIN. >7 log-kill and >5 log-kill were acheived vs. vegetative microbes (<30 sec) and spores (60 sec), respectively, under optimized variables. Presence of UV light and sample distance from and time under the device were the most important variables. 254 nm UV had a significantly better AME than 222 nm; combining both UV lamps had a significant synergistic AME. The narrowest UV beam (2 mm) yielded the greatest AME (total energy input kept constant). Adding O3 to UV had a modest but significant synergistic effect; optimal [O3] was 0.3-0.8 ppm. Changing airflow velocity had no significant effect on AME. Conclusion HyLuxO3 is a novel device that achieves >7 log-kill vs. common pathogenic vegetative microbes and >5 log-kill vs. spores using combined UV light and [O3] safe for human skin antisepsis (and surface/fomite decontamination)- and- yields such impressive AME on faster timescales than those required by bleach/other chemical products unsuitable for human skin. Future studies on human hands (using many other microbes) will determine if HyLuxO3 meets regulatory and efficacy requirements for use in and beyond healthcare settings, especially with the specter of emerging respiratory viruses.