Disinfection of microenvironment surface is very important for control contamination of biosafety laboratory. Hydrogen peroxide has been used widely in biosafety laboratories for disinfection. The effect of hydrogen peroxide on inactivation of different bacteria was investigated in the present study. The effect of hydrogen peroxide in the form of dry-mist (diameter <10 µm) on sterilization of common pathogens in the biosafety cabinet (BSC) was evaluated. The optimized procedure (diffusing 1 min for 8 cycles, reaching 60 ppm, and staying 2 h) has been determined, at which the hydrogen peroxide dry-mist was able to completely kill 6 logs of Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Mycobacterium smegmatis (M. smegmatis), Bacillus subtilis (B. subtilis) and Bacillus stearothermophilus (B. stearothermophilus), as well as 8 logs of Escherichia coli (E. coli), but not 7 logs of S. aureus, S. epidermidis and M. smegmatis. Therefore, it is suggested that before disinfection of BSC surface microenvironment by hydrogen peroxide dry-mist it is necessary to wipe the surface of BSC or decontaminate extravasation spilth with disinfectants.