The objective of the investigations was to determine the viability of selected gram-negative bacteria exposed to high pressure and sub-zero temperature, in the range of 59-193 MPa and –5 - –20°C, respectively, without the freezing of water. After pressurization for 24 h, at 193 MPa and –20°C, no living cells of gram-negative bacteria T. thermophilus, P. fluorescens and E. coli of the initial population number of 10⁸ CFU/mL were detected. After pressure treatment for a shorter time than 24 h, differences in sensitivity appeared between these bacteria. The most resistant was the tested strain of E. coli. Reduction in the population of E. coli was only about 3.5 log cycles after pressurization for 30 min at –20°C, while T. thermophilus and P. fluorescens were completely inactivated under these conditions. No living cells of E. coli were found after 7 h of pressure treatment at 193 MPa and –20°C. The viability of all tested bacteria was not reduced significantly at 59 MPa, but decreased with the pressure increase. The most sensitive was P. fluorescens - a drastic loss of viability in these bacteria occurred during the time of generation of the pressure of 110 MPa at –10°C. In this period, the reduction in viable numbers of E. coli did not exceed 1 log cycle in all studied ranges of pressure and temperature. Freezing the samples under atmospheric pressure at –5°C - –20°C did not exert any influence on the viability of E. coli and T. thermophilus, while the number of viable cells of P. fluorescens decreased by 0.5 - 1 log cycle.