Cell suspensions of Listeria monocytogenes 5366, Staphylococcus aureus 976 and Pseudomonas aeruginosa 110T were exposed to high pressure treatment from 200 to 600 MPa for 5, 10 and 15 min at an initial temperature of 20 °C in raw skimmed bovine milk, whey and phosphate buffer, pH 7.0 in order to determine their degree of inactivation. S. aureus was the most baroresistant bacteria followed by L. monocytogenes and P. aeruginosa. A treatment at 600 MPa for 15 min was needed to obtain a reduction of 7 log10 cycles of S. aureus in all media. This treatment was selected to determine the degree of denaturation of the main whey proteins. Lactoperoxidase activity was not affected by pressure treatment applied to milk, whereas a denaturation of 80%, 70%, 44% and 7% was obtained for β-lactoglobulin, lactoferrin, immunoglobulin G (IgG) and α-lactalbumin, respectively. The effect of the treatment media on denaturation was not noticeable for lactoperoxidase, α-lactalbumin and IgG whereas β-lactoglobulin was more sensitive when treated in milk and lactoferrin in whey. Treatment of milk at 75 °C for 15 s induced a lower denaturation of β-lactoglobulin, lactoferrin and IgG compared to pressure treatment, whereas produced a higher decrease of enzymatic activity of lactoperoxidase.