High pressure treatments at 400, 500, 550, and 600 MPa for 15–30 min at room temperature were applied to milk inoculated with a single strain of Gram-positive (Listeria monocytogenes and Staphylococcus aureus) or Gram-negative (Escherichia coli) bacteria or their mixture. This study showed that the high pressure-induced inactivation of microorganisms depends on such factors as microbial species, pressure, concentration of the inoculant, and presence of other bacterial species. There were significant (p < 0.05) differences in the inactivation rate of bacteria among pressure treatments. E. coli ATCC 25922 showed lower pressure resistance than L. monocytogenes ATCC 7644 and S. aureus ATCC 25923. S. aureus is more resistant in a mixture; recovery of S. aureus was detected after 8 days for a single inoculum and 1 day in a mixture of milk storage at 4 ± 1 °C.