Dimethyldioxirane (DMDO), generated in situ by adding acetone to an aqueous solution containing potassium peroxymonosulfate (Oxone) at neutral pH, was investigated for inactivation of biological warfare agent simulants. The DMDO solution inactivated bacterial spores, fungal spores, vegetative bacterial cells, viruses, and protein by 7 orders of magnitude in less than 10 min. The kill rates of DMDO were more pronounced when compared to kill rates of buffered Oxone alone. Conditions for the use of DMDO as a biological decontaminant were optimized by evaluating the effects of age and temperature on open systems. DMDO effectiveness was compared to that of current decontaminant solutions such as DS2 (used by the U.S. military), bleach, and hydrogen peroxide and was shown to be superior in achieving a 7-log kill of Bacillus atrophaeus, a Bacillus anthracis spore simulant. The results demonstrate the potential for DMDO to fill the need for a noncorrosive, nontoxic, and environmentally safe decontaminant.