The purpose of this study was to evaluate the synergistic bactericidal efficacy of combining ultrasound (US) and fumaric acid (FA) treatment against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes in apple juice and to identify the synergistic bactericidal mechanisms. Additionally, the effect of combination treatment on juice quality was determined by measuring the changes in color, pH, non-enzymatic browning index, and total phenolic content. A mixed cocktail of the three pathogens was inoculated into apple juice, followed by treatment with US (40 kHz) alone, FA (0.05, 0.1, and 0.15%) alone, and a combination of US and FA for 1, 2, 3, 4, and 5 min. Combined US and 0.15% FA treatment for 5 min achieved 5.67, 6.35, and 3.47 log reductions in E. coli O157:H7, S. Typhimurium, and L. monocytogenes, respectively, with the 1.55, 2.37, and 0.57 log CFU reductions attributed to the synergistic effect. Although the pH value slightly decreased as FA increased, there were no significant (P > 0.05) differences in color values, browning indices, and phenolic content between untreated and treated samples. To identify the mechanism of this synergistic bactericidal action, membrane integrity, malfunctions in the membrane efflux pump, and intracellular enzyme activity were measured. The analyses confirmed that damage to the cell envelope (membrane integrity and efflux pump) was strongly related to the synergistic microbial inactivation. These results suggest that simultaneous application of US treatment and FA is a novel method for ensuring the microbial safety of apple juice.