We investigated the effect of carbonation treatment with heating (CH) in the presence of glycerin fatty acid esters on Bacillus subtilis spores. The inactivation effect of CH was the highest in the presence of monoglycerol monocaprate (MC10). Monoglycerol monolaurate showed the highest bacteriostatic effect. The resistance of spores to CH with MC10 did not change with pre-heat activation. Freshly prepared spores from the surviving spores following CH with MC10 showed resistance similar to that of normal spores. The resistance of spores might be affected by the composition of the spore-forming medium. CH with MC10 enhanced adhesion of MC10 to spores and dipicolinic acid (DPA) release from spores, but did not induce DNA fragmentation. Inactivation of B. subtilis spores by CH with MC10 may be attributable to the impairment of physiological germination and/or decrease in heat resistance, which were induced by enhanced MC10 adhesion and the resultant DPA release.