Mycobacterium tuberculosis is an important human pathogen that is routinely cultured in clinical and research laboratories. M. tuberculosis can contaminate surfaces and is highly resistant to disinfection. We investigated whether hydrogen peroxide vapor (HPV) is effective for the deactivation of M. tuberculosis on experimentally contaminated surfaces in a biological safety cabinet (BSC) and a room. Biological indicators (BIs) consisting of an approximately 3-log(10) inoculum of M. tuberculosis on stainless steel discs and a 6-log(10) inoculum of Geobacillus stearothermophilus were exposed to HPV in BSC time course experiments and at 10 locations during room experiments. In three separate BSC experiments, M. tuberculosis BIs were transferred to growth media at 15-min intervals during a 180-min HPV exposure period. No M. tuberculosis BIs grew following 30 min of HPV exposure. In three separate room experiments, M. tuberculosis and G. stearothermophilus BIs were exposed to HPV for 90, 120, and 150 min, respectively. BIs for both microorganisms were deactivated in all 10 locations following 90 min of HPV exposure. HPV provides an alternative to traditional decontamination methods, such as formaldehyde fumigation, for laboratories and other areas contaminated with M. tuberculosis.