To improve the inactivation efficiency of low pressure CO2 microbubbles (MB-CO2), we developed the MB-CO2 equipment from batch system to two-stage MB-CO2 system which was additionally pressurized at ambient temperature after MB-CO2 was mixed with a solution at lower temperature and pressure. Therefore, the effect of the two-stage MB-CO2 treatment for inactivating Saccharomyces cerevisiae was investigated. The two-stage MB-CO2 efficiency for the inactivation of S.cerevisiae dramatically increased by lowering the temperature and by increasing the pressure in the mixing vessel. Furthermore, its effectiveness significantly enhanced by increasing the temperature in the heating coil, although not by the pressure. As a result, using the two-stage MB-CO2 equipment was suggested as a new effective technique to inactivate S.cerevisiae. © 2013 Elsevier Ltd.