The extreme resistance of bacterial spores to most killing agents makes them a major concern to the food industry and consumers. This gave rise an increasing interest in developing new strategies to inactivate spores and understand the mechanisms of inactivation by various agents. In this study, ultrasound combined with heat (thermosonication, TS) was used to inactivate the spores of Bacillus subtilis and the factors that influence the resistance to TS were analyzed. The spores of wild-type B. subtilis and isogenic mutants were subjected to heat at 80 °C and ultrasound at 6.67-20 W/mL and 23 °C for 0-40 min. TS treatment has synergistically resulted in spore inactivation, and spores of wild-type B. subtilis and isogenic mutants showed different resistance to TS treatment, which was in the following order: Strains 533 (wild-type) ≈ strains PS3518 (gfp) ≈ strains PS2318 (recA-) > strains PS578 (α-β-), and spores of strains PS3328 (cotE-) were also more susceptible than those of wild-type strains. The inactivated spores lost some proteins in the spore core but initiated germination normally. The germinated inactivated spores did not swell and their plasma membrane permeability was equally altered. It was concluded that the damage to spores' inner membrane (IM) proteins or the IM itself has led to the leakage of intracellular substances and the death of a spore by TS treatment. Our results could support the development and optimization of TS treatment, which has great significance for its further utilization in food industry.