Abstract: The antibacterial activity and antibacterial mechanism of the eucalyptus oil on the four microorganism of aquatic products in vitro was investigated in the study. Firstly, the antibacterial activity of four selected EOs including tea tree oil, eucalyptus oil, clove oil and mentha oil against four tested bacterial strains including Escherichia coli, Shewanella putrefaciens, Vibrio parahaemolyticus and Staphylococcus aureus were tested by means of agar diffusion and broth dilution methods, respectively. In which, the diameters of inhibition zone (DIZ) and minimal inhibitory concentration (MIC) were tested to evaluate the antibacterial activity of the EOs respectively. Furthermore, the antibacterial mechanism of eucalyptus oil against the model bacteria including Gram-negative S. putrefaciens and Gram-positive S. aureus was deeply investigated. In the mechanism researching experiment, scanning electron microscopy (SEM), transmission electron microscopy (TEM), the electric conductivity, nucleic acid content and the intracellular ATP content at MIC were tested to determine cell membrane permeability, membrane integrity and cell physiological function, respectively. The results showed that the diameters of inhibition zone of eucalyptus oil against Escherichia coli, Shewanella putrefaciens, Vibrio parahaemolyticus and Staphylococcus aureus were 33.33, 27.87, 38.88, 39.61 mm, respectively. The MIC values of Escherichia coli, Shewanella putrefaciens, Vibrio parahaemolyticus and Staphylococcus aureus were 1.00, 1.25, 0.75, 0.63 μL/mL, respectively. Eucalyptus oil performed the strongest antibacterial performance to all tested strains The bacteria cells morphology and ultrastructure were apparently damaged by eucalyptus oil, the electric conductivity and the optical density (OD₂₆₀) value of the treated bacterial increased, meanwhile, the intracellular ATP content of the treated bacterial decreased. It could be concluded that the bacteria cells membrane permeability, integrity and cell physiological function were damaged when bacteria cells were exposed to the eucalyptus oil at MIC. From the study referred above, it was demonstrated that the cell membrane was affected by eucalyptus oil, resulting in a loss of cell homeostasis. The experiment will provide a theoretical basis for the application of eucalyptus oil as a natural antibacterial substance in aquatic products industry.