This study evaluated the synergistic antibacterial mechanism of ultrasound (US) and rosmarinic acid (RA) treatment on large yellow croaker during cold storage (4 °C). The results indicated that the synergistic treatment of US + RA can significantly reduce TVB-N and TCA-soluble peptide and decrease protein decomposition and oxidation in large yellow croaker during storage, and thus significantly delay the growth of microorganisms. In addition, the representative strains of Shewanella balticaand Carnobacterium maltaromaticumwere isolated from the decaying yellow croaker, and the antibacterial mechanism of the combination of US + RA was determined. Compared with Carnobacterium maltaromaticum, the synergistic treatment of US + RA has a more significant impact on the membrane structure of the Shewanella baltica, which exacerbates the leakage of nucleic acid and protein in the Shewanella balticaand effectively inhibits its respiratory metabolic activity. This result provides evidence for the antibacterial mechanism and preservation ability of the combination of US + RA.