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The effective use of disinfection and sterilization is important in preventing healthcare associated infections. Especially, multi-drug resistant organism and spore-forming bacteria are serious pathogens. Therefore, control of a nosocomial infection is emerging as a big problem. It is efficiently necessary to eliminate the pathogen. In this study, the components of sterilant were optimized. The optimized sterilant was applied for efficiency of sterilization using G. stearothermophilus E. coli, S. aureus as a standard microorganism. Our results were shown as follows : The decomposition or loss of potency of sterilant in small containers did not appeared for one month at ambient temperature. All of hydrogen peroxide 0.5%, peracetic acid 0.01%, and IPA 60% was effective for sterilization of E. coli, S. aureus using dilution method. Two kinds of sterilants(hydrogen peroxide 2%+peracetic acid 0.1%+IPA 0.5%, hydrogen peroxide 4%+peracetic acid 0.06%+IPA 1%) were bacteriocidal(> 6 log reduction). Microorganisms(E. coli, S. aureus) which treated with these sterilants for 2min were not grown in LB broth at 7 days culture. The gas plasma system for sterilization was applied. The sterilant solution(hydrogen peroxide 20%+IPA 5%) was vaporized in the sterilization chamber. The vaporized sterilant was bacteriocidal and sporocidal(> 6 log reduction) using G. stearothermophilus E. coli, S. aureus as biological indicators.