Chitosan, a natural biopolymer with antibacterial, antibiofilm, antifungal and antiviral activity, is a renewable resource that can be obtained plenty from natural resources, is environmental friendly non-toxic to living things, has high biodegradability, biocompatibility and usability in many forms. In this study, was aimed to determine its antibiofilm activity on Escherichia coli biofilms of chitosan. For this purpose both E. coli planktonic cells and on E. coli biofilm of the chitosan was respectively detected inhibition effect and cell viability by XTT reduction assay. Then at different times (8 and 24 hours) were imaged structural differences between biofilm formation by Scanning Electron Microscope. Furthermore the exopolysaccharide production of this bacteria, in presence of the chitosan has been identified. It has been found out that the chitosan with a concentration of 0.125% was inhibited more than 50% of the cells in the 8 and 24 hours development phases. In the presence of the same concentrations of chitosan was determined that both are distribute exopolymeric matrix on the biofilm surface and lowest exopolysaccharide production occurs. Therefore, chitosan may proposed to be a antibiofilm agent which is effective on E. coli biofilms, but further in vivo studies should be conducted.