This study assessed the efficacy of gaseous chlorine dioxide for inactivation of Bacillus anthracis, Francisella tularensis, and Yersinia pestis in a hospital patient care suite. Spore and vegetative cells of Bacillus anthracis Sterne 34F2, spores of Bacillus atrophaeus ATCC 9372 and vegetative cells of both Francisella tularensis ATCC 6223 and Yersinia pestis A1122 were exposed to gaseous chlorine dioxide in a patient care suite. Organism inactivation was then assessed by log reduction in viable organisms postexposure to chlorine dioxide gas compared to non-exposed control organism. Hospital room decontamination protocols utilizing chlorine dioxide gas concentrations of 377 to 385 ppm maintained to exposures of 767 ppm-hours with 65% relative humidity consistently achieved complete inactivation of B. anthracis and B. atrophaeus spores, as well as vegetative cells of B. anthracis, F. tularensis, and Y. pestis. Decrease in exposure (ppm-hours) and relative humidity (<65%) or restricting airflow reduced inactivation but achieved >8 log reductions in organisms. Up to 10-log reductions were achieved in a hospital room with limited impact on adjacent areas, indicating chlorine dioxide concentrations needed for decontamination of highly concentrated (>6 logs) organisms can be achieved throughout a hospital room. This study translates laboratory chlorine dioxide fumigation studies applied in a complex clinical environment.