Mycobacterium tuberculosis is a major cause of morbidity and mortality worldwide and is becoming a greater concern due to the development of multidrug-resistant strains. M. tuberculosis can contaminate rooms, medical equipment, and research laboratories and has the propensity to be highly resistant to decontamination. The aim of this study was to determine the efficacy of room disinfection with a dry mist of hydrogen peroxide (DMHP) in a biosafety level 3 laboratory in the event of contamination with M. tuberculosis. The biological indicators (BIs) were comprised of presterilized cotton tissues on which amounts of about 10(7) CFU/ml of M. tuberculosis H37Ra were dried. The device (Sterinis; Gloster Sante Europe) provided a DMHP of 5% hydrogen peroxide during 25 min. Three experiments were performed. The viable bacteria were reduced by values of more than 5 log(10), and no colony grew from any BI. In conclusion, DMHP shows promise as an effective and safe alternative to the currently used formaldehyde.