OBJECTIVE We evaluated gaseous chlorine dioxide (ClO2) decontamination of an ambulance using a variety of bacterial biological agents. METHODS Spores of attenuated Bacillus anthracis and Bacillus atrophaeus as well as vegetative cells of Acinetobacter baumannii, Mycobacterium smegmatis, and Staphylococcus aureus were exposed to ClO2 gas inside an ambulance. Log reduction in viability was assessed following decontamination using organism plate counts. RESULTS Ambulance decontamination with ClO2 gas concentrations of 362 to 695 ppm maintained to exposures of 756 ppm-hours with 65% relative humidity (RH) achieved inactivation of all the bacterial agents tested. Decreasing exposure (ppm-hours) and RH (<65%) or restricting air flow reduced inactivation but still achieved greater than 6-log reductions in organism viability. CONCLUSIONS Up to 10-log reductions were achieved in an ambulance interior following exposure to ClO2, indicating that gas concentrations needed to mitigate biological agent contamination can be achieved and maintained safely in an ambulance. Future studies are ongoing to evaluate gaseous ClO2 in other environments contaminated with biological agents of health care concern.