The synergetic effect of nisin and high-pressure carbon dioxide (HPCD) on structural changes and leakage of nucleic acid, soluble proteins, Mg²⁺, Ca²⁺, K⁺, and dipicolinic acid from Bacillus subtilis spores was studied. Both HPCD and nisin auxiliary were used to inactivate the B. subtilis spores. The death rate of spores treated by HPCD combined with the nisin were significantly higher than HPCD alone (P < 0.05). The main cause of spore’s death is the leakage of components caused by the change of spore permeability and structural damage. The HPCD treatment damaged the spore ultrastructure, resulting in the leakage of nucleic acid, Mg²⁺, Ca²⁺, K⁺, and dipicolinic acid from spores, while nisin auxiliary to HPCD treatment damaged spore membrane, which led to spore death and play a synergetic effect. This study evaluated synergetic effects of HPCD combined with the bacteriocin nisin. The investigation provided evidence for potentially combined application of HPCD and nisin to help ensure food safe in the industry.