A high-level aseptic environment must be maintained in bio-cleanrooms used for the manufacture of sterile products. In the past, formaldehyde gas was most commonly used to sterilize bio-cleanrooms, but due to strict residual limitations there has been a need to develop a less toxic alternative choice. The authors have developed a revolutionary new sterilization system using a high concentration of ozone gas and used this system to sterilize an actual bio-cleanroom. This system integrates the ozone gas generator with the air conditioning system by proper control. The design specifications for the system included an ozone gas concentration of 200ppm or more, relative humidity of 80% or more, and a sterilizing time of 120min. Blow vents and suction ports were placed to ensure a uniform airflow which would extend through the entire room during ozone gas sterilization. Tests regarding long-term material exposure to ozone gas were conducted when the system was introduced to distinguish usable and unusable materials. In an actually constructed cleanroom, simulations were used to predict the evenness of the diffusion of ozone gas concentration and relative humidity during ozone gas sterilization, and measurements of the actual indoor ozone gas concentration, temperature and relative humidity during sterilization revealed that the ozone concentration and relative humidity needed for sterilization had been achieved generally throughout the entire environment. In addition, the CT value (mg/m³ (=ppm) ×min) , derived by multiplying the ozone gas concentration during ozone gas sterilization by the sterilization time, was equal to or greater than the target value of 24×10³ (ppm·min) . When the results of sterilization in a cleanroom were confirmed using a biological indicator (BI) , negative results were obtained at all measurement points, demonstrating that sterilization was being performed effectively in the actual factory at which the ozone gas sterilization system had been introduced.