The mechanisms of ions retained in biofilm matrices were investigated and clarified in this study. Biofilm polymer characterization was conducted by measuring the electrical charge mobility and the potentiometric titration. The ion retained mechanism was explored in more detail by investigating the ion adsorption and desorption mechanisms of the biofilm matrices. The results suggested that both positively and negatively charged sites exist in the biofilm polymers. A physicochemical interaction between the sites and the ions in the surrounding water seems to promote the ion adsorption to the biofilm through an attractive electrostatic interaction and an ion-exchange mechanism. The attracted ions may be reserved in two regions; 1) the regions between biofilm polymers and 2) on the charged sites of biofilm polymers. The main factor regulating the ion retaining ability inside of biofilms seems to be an electrostatic interaction between the ions and the charged sites of biofilm polymer. The greater attractive electrostatic attraction between the retained ions and the charged sites of biofilm polymers will hinder the release of the ions from the biofilm matrices. The result obtained by the present study enables us to find out the determinant factor regulating the ion retained mechanisms of the natural biofilm matrices.