Abstract The antimicrobial mechanism of Ginkgo biloba leaf extracts (GBLE) when applied to predominant spoilage bacteria (Shewanella putrefaciens and Saprophytic staphylococcus) on refrigerated pomfret and minimal inhibitory concentrations (MICs) were measured by the plate counting method. GBLE at MIC and 2MIC were prepared in tryptic soy broth (TSB) medium and equivalent amounts of sterile distilled water were used in place of GBLE as a control group. The impact of GBLE on the growth of bacteria, the permeability of cell membrane, and cell wall were also investigated by growth curve of bacteria, alkaline phosphates activity (AKP), and electrical conductivity. A scanning electron microscope (SEM) was used to study the effects of GBLE on the cellular structure of S. putrefaciens and S. staphylococcus. The results showed that the MICs of GBLE when applied to S. putrefaciens and S. staphylococcus were 100 mg/mL, the inhibitory rates of MIC and 2MIC concentrations of GBLE when applied to S. putrefaciens were 36.11% and 100%, while 27.78% and 62.22% for S. staphylococcus. Meanwhile, GBLE inhibited the growth of S. putrefaciens and S. staphylococcus until the number of cells at 2MIC values decreased to 0 and 4.29 log CFU/mL, respectively, after 24 h. The electrical conductivity of bacteria increased with GBLE treatment, which was followed by an increased leakage of AKP. The SEM revealed that the structure of bacterial cells was destroyed and the bacteria began to be adhere to each other. The inhibition effect of GBLE when applied to S. putrefaciens and S. staphylococcus was related to the damage of cell membrane and cell wall. It was also revealed that GBLE damages the morphology of bacteria and had stronger effects on the cell membrane of S. putrefaciens than that of S. staphylococcus.