The sterilization of transplant and medical devices should be effective but not detrimental to the structural properties of the materials used. In this study, we examined the effectiveness of chemical and physical agents for inactivating Staphylococcus aureus, a gram-positive bacterium and important cause of infections and biofilm production. The treatment conditions in this work were chosen to facilitate their subsequent use with sensitive materials. The effects of temperature, high hydrostatic pressure, and glutaraldehyde disinfectant on the growth of two strains of S. aureus (ATCC 25923 and BEC 9393) were investigated individually and/or in combinations. A low concentration of glutaraldehyde (0.5 mM), high hydrostatic pressure (300 MPa for 10 min), and moderate temperature (50 degrees C), when used in combination, significantly potentiated the inactivation of both bacterial strains by > 8 orders of magnitude. Transmission electron microscopy revealed structural damage and changes in area that correlated with the use of pressure in the presence of glutaraldehyde at room temperature in both strains. Biofilm from strain ATCC 25923 was particularly susceptible to inactivation. The conditions used here provided effective sterilization that can be applied to sensitive surgical devices and biomaterials, with negligible damage. The use of this experimental approach to investigate other pathogens could lead to the adoption of this procedure for sterilizing sensitive materials.