Bacillus cereus is a typical spore-forming strain that can transform into dormant spores to resist unfavorable growth conditions. Among the microorganisms that cause spoilage of dairy products, B. cereus accounts for a large proportion. In this study, we evaluated the inhibitory activity of monolauroyl-galactosylglycerol (MLGG) on B. cereus spores and explored its inhibitory mechanism. The minimum inhibitory spore concentration (MISC) and the minimum sporicidal concentration (MSC) of MLGG against B. cereus spores were 0.313 mg/mL and 2.5 mg/mL, respectively. Scanning electron microscopy was used to observe the changes in spore morphology after MLGG treatment. It was found that low concentrations of MLGG caused irregular spore shape, with a few spores slightly ruptured and high concentrations of MLGG increased the degree of spore rupture, resulting in significant leakage of spore contents. By measuring the effect of MLGG on spore structure, it could be determined that MLGG damaged the coat of the spores and weakened the resistance of the spores, but did not affect the activity of cortical lyases. Therefore, spores could not resume growth even with the addition of exogenous lytic enzymes. After propidium iodide (PI) staining of the spores, flow cytometry analysis revealed that the positive area gradually increased, indicating that the permeability of the inner membrane of the spores was disrupted, leading to dipicolinic acid (DPA) release. The amount of DPA release was proportional to the MLGG concentration, and the growth rate of the spores decreased under hypertonic conditions. These findings indicated that MLGG inactivated spores primarily by damaging the inner membrane of the spores.