Abstract: In this study,the inhibitory effect of thymol on E.cloacae C4 biofilm formation at sub-inhibitory concentration was studied. Firstly the minimal inhibitory concentration(MIC)of thymol on E.cloacae C4 was determined. Then the effect of sub-inhibitory concentration of thymol on the bacterial counts in the biofilm and themobile ability of the cells and the content of exopolysaccharides were determined. Changes in bacteria morphology and three-dimensional structure of biofilm were observed. The relative expression of genes associated with biofilm formation were determined. The results indicated that the MIC of thymol was 256 μg/mL. 1/4MIC of thymol was not affect the growth of planktonic bacteria and the bacterial counts in biofilm but significantly(P<0.05)inhibited the mobile ability of bacteria and the content of exopolysaccharides.The expression of genes related to biofilm formation(cell adhesion and the synthesis of exepolysaccharides)was down-regulated. The biofilm bacteria morphology grew longer,the biofilm thickness decreased and the membrane structure was damaged after thymol treatment. Therefore,thymol has an inhibitory effect on the biofilm formation of E.cloacae C4 under the sub-inhibitory concentration.