High-pressure homogenization has been extensively studied for its excellent homogenization effect and the prospect of continuous liquid food production, but its sterilization ability still needs to be improved. In this study, we replaced the homogenization valve with two opposing diamond nozzles (0.05 mm inner diameter) so that the fluid collided at high velocity, corresponding to high-pressure micro-fluidization (HPM). Moreover, HPM treatment significantly inactivated Staphylococcus aureus ~7 log in the liquid with no detectable sub-lethal state at a pressure of 400 MPa and a discharge temperature of 50 °C. The sterilization effect of HPM on S. aureus subsp. aureus was attributed to a significantly disrupted cell structure and increased membrane permeability, which led to the leakage of intracellular proteins, resulting in bacterial death. At the same time, HPM treatment was able to significantly reduce the ability of S. aureus subsp. aureus to form biofilms, which, in turn, reduced its virulence. Finally, compared to the simulated system, more effective sterilization was observed in apple juice, with its color and pH remaining unchanged, which suggested that HPM can be used to process other liquid foods.