This study evaluated the inactivation effect of ultrasonic treatment combined with acidic electrolyzed water (AEW) on Bacillus cereus spores. AEW treatment reduced the spores by 1.05-1.37 log CFU/mL while the sporicidal effect of ultrasound was minor. More strikingly, simultaneous ultrasonic and AEW treatments for 30 min led to 2.29 log CFU/mL reduction and thus, considered a synergistic effect. Flow cytometry combined with SYTO/PI staining analysis revealed that ultrasound hydrolyzed the cortex while the AEW partially damaged the integrity of the inner membrane. Scanning and transmission electron microscopies were used to characterize the ultrastructural changes. The detachment of the exosporium induced by ultrasound was the most apparent difference compared with the control group, and the electron density of spores appeared to be heterogeneous after treatment with AEW. These results indicated that combining ultrasound with AEW is a promising decontamination technology with potential uses in the food industry and environmental remediation.