The synergistic effect of frozen-phase high pressure (HP) on the inactivation of E. coli ATCC 25922 cultures in suspension medium, Chinese bayberry juice (pH 3.0), and carrot juice (pH 6.5) was evaluated. The survivor count of E. coli remained at 3.36 log CFU/mL on a nonselective brain heart infusion (BHIA) medium, while no survivor was detected on a selective violet red bile agar (VRBA) medium after a 5 min hold pressure at 250 MPa in a frozen culture suspension. BHIA was suitable for safe testing of the injured E coli cells after HP treatment in frozen state. Frozen Chinese bayberry juice showed higher sensitivity to HP treatment for its matrix property with high sterilizing efficiency at 170 MPa. Two pulses exhibited a significant inactivation effect in frozen samples compared with one pulse, especially for the Chinese bayberry juice with different pressure levels. The destruction kinetics of HP pulse mode followed the first-order rate kinetics with a Zp value of 267 MPa in frozen carrot juice. Our results evaluated the influenced factors of frozen HP destruction effects, including the medium, substrate, and application mode. The frozen HP destruction kinetics of pulses afford us better understanding of the technology application in the food industry.