This study evaluated the inactivation of Brucella suis, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis on glass, Hypalon® rubber glove, and stainless steel using vaporous hydrogen peroxide fumigation of a ∼15 m3 chamber. A suspension of approximately 1 × 108 colony forming units (CFU) of each organism was dried on coupons of each type of test surface and exposed to vaporous hydrogen peroxide. A significant reduction in the log10 CFU of each organism on all test materials was observed between the controls evaluated after a 1-hour drying time and unexposed controls evaluated after decontamination. For all organisms, qualitative growth assessments showed that vaporous hydrogen peroxide exposure completely inactivated bacterial viability on all replicates of the test materials incubated up to 7 days post-exposure. In parallel, all Geobacillus stearothermophilus biological indicators (BI) exposed to vaporous hydrogen peroxide exhibited no growth after 1 and 7 days incubation. This study provides information on using a combination of quantitative and qualitative growth assessments to evaluate vaporous hydrogen peroxide for the surface decontamination of B. suis, B. pseudomallei, F. tularensis, and Y. pestis within a large-scale chamber.