Environmental disinfection in a health care setting is an important aspect of infection control. Recently, there has been interest in the use of vapor- and gas-based treatments for decontamination of surfaces and rooms. We describe preliminary results for an ozone-based decontamination of surfaces seeded with a range of vegetative cells and spores of bacteria of clinical relevance. The efficacy of the approach for room sanitization was also assessed. The protocol included use of a quenching agent to rapidly reduce ozone concentrations to safe levels allowing treatment times of less than 1 h for the majority of organisms tested. Using bacteria seeded onto agar plates and solid surfaces, reductions in bacterial load of greater than 3 log values were recorded for a number of organisms including Escherichia coli and methicillin-resistant Staphylococcus aureus . Application of the process in a 30 m³room showed similar reductions in viable counts for these organisms and for Clostridium difficile spores. We suggest that the potential of this ozone–quench approach should be further evaluated for disinfection or decontamination of healthcare environments.