Bacterial spores are common microbes in foods. They are difficult to be inactivated because of their extreme resistances. The synergistic inactivation effects and mechanism of Nisin combined with ε-polylysine treatment on Bacillus subtilis spores in thermal physical field (80, 90 ℃) were studied, and a more effective method for food preservation was provided. The changes of OD_{600 nm}, OD_{260 nm}, OD_{280 nm} values were used for characterizing the inner membrane damage and intracellular components release. Scanning electron microscope (SEM) was used for observing the changes in spore morphology and structure. Fourier transform infrared (FTIR) spectroscopy was used for analyzing the changes in lipids phase of spore inner membrane, the secondary structure of spore proteins, and nucleic acid backbone. The activity of Na⁺/K⁺-ATPase was determined to evaluate spore's metabolic condition. The results are as follows: Under 90 ℃ treatment, 1.59 lg CFU/mL or 1.22 lg CFU/mL Bacillus subtilis spores were inactivated respectively with addition of 0.5 g/L Nisin or 0.25 g/L ε-polylysine, while 4.28 lg CFU/mL spores were inactivated with combined usage of 0.5 g/L Nisin and 0.25 g/L ε-polylysine. After the combined treatment, SEM images indicated that the aggregation of spores significantly intensified. The 2,6-dipicolinic acid (DPA) release raised up to 76.03%, indicating serious inner membrane damage. FTIR spectroscopy indicated that the lipids in the spore inner membrane changed from a gel state to a liquid crystal state, nucleic acid backbone was damaged, and the content of secondary structures in proteins changed (P<0.05), the α-helix and β-folding content decreased, while the β-turn and random coil content increased respectively to 43.81% and 35.37%. In addition, the activity of Na⁺/K⁺-ATPase significantly decreased to lowest value 6.53 μmol/h/mg (P<0.05), and the energy metabolism of spores was severely inhibited. The main mechanism of synergistic spore inactivation by Nisin and ε-polylysine was as follows: Nisin destroyed the inner membrane structure of spores, which resulted in core hydration and release of key components such as DPA. ε-Polylysine penetrated the damaged inner membrane, entered the spore core, destroyed DNA structure, and finally caused spore inactivation.