The main objective of this study was to assess the in vitro antibacterial effectiveness ofOcimum gratissimumL. essential oil (OGEO) against Shewanella putrefaciens. The minimum inhibitory concentration and minimum bactericidal concentration of OGEO acting on S. putrefaciens were both 0.1% and OGEO could inhibit the growth of S. putrefaciens in a dose-dependent manner. The restraint of the biofilm growth of S. putrefaciens was found in the crystal violet attachment assay and confocal laser scanning microscopy. The disruption of cell membranes and exudation of contents in S. putrefaciens with OGEO treatment were observed by scanning electron microscopy, hemolysis and ATPase activity. The results demonstrated that OGEO had a positive inhibitory effect on the growth of S. putrefaciens, which primarily developed its antibacterial function against S. putrefaciens by disrupting the formation of biofilms and cell membranes. This study could provide a new method of inhibiting the spoilage of food in which the dominant spoilage bacteria are S. putrefaciens.