Bacillus subtilis and Escherichia coli were chosen to investigate the combined effect of high hydrostatic pressure (HHP) and Nisin on loss of viability, membrane damage and release of intracellular contents of microorganisms. The results showed that the combination of 200 IU/mL Nisin and HHP exhibited a synergistic effect over 2 log on the inactivation of B. subtilis at pressure 300 MPa. The similar synergistic effect was observed on the membrane damage and release of intracellular contents of B. subtilis. The Nisin alone had no effect against E. coli, which belongs to gram negative bacteria. However, at pressure 300 MPa, Nisin caused the membrane damage from 55% to 80%. The synergistic effect of Nisin and HHP on loss of viability, membrane damage and release of intracellular contents of E. coli were also illustrated when the HHP pressure exceeded 300 MPa as the consequence of the serious changes produced by HHP at higher pressure in the cell envelope. It allows the entry of Nisin molecules to cell membrane.