Shewanella putrefaciens and Listeria monocytogenes are concerned by scientists and consumers due to their ability to produce dense biofilms to contaminate and cause food spoilage. The object of this study was to investigate the antimicrobial and anti-biofilm activities of octyl gallate (OG) against the two food-related bacteria. Results showed that the antimicrobial activities of OG were in a dose-dependent manner and the minimum inhibitory concentration (MIC) of S. putrefaciens and L. monocytogenes were 0.2 mmol/L and 0.1 mmol/L, respectively. OG can rapidly penetrate the bacteria and produce reactive oxygen species (ROS). Then, OG and ROS can lead to membrane damage and cell rupture, causing DNA and protein leakage. Our data also strongly suggested that OG not only can inhibit biofilms formation, but also eradicate the performed biofilms. Furthermore, it was shown that OG, as an additive, has great potential in antimicrobial application to prolong the shelf life of large yellow croakers. These prove that OG can combat bacteria and eradicate biofilm against S. putrefaciens and L. monocytogenes.