The electrochemical impedance spectroscopy (EIS) technique has been used as a sensitive method to explore the effect of antibacterial molecules on immobilized bacteria and biofilm formation. In this work, we describe the electrochemical spectroscopy as a powerful method to monitor the effect of Chlorhexidine Digluconate (CHX-Dg) on polyelectrolyte immobilized Escherichia coli K12 MG1655 and the kinetics of cell adhesion on gold electrodes. The experimental impedance data were modelised with a Zview program to find the best equivalent electrical circuit and analyse its parameter's properties. Polyelectrolyte multilayer formation on the electrode surface and bacteria immobilization greatly increased the electron-transfer resistance (Ret) and reduced the constant phase element (CPEdl). The effect of CHX-Dg was studied in a 0.5 × 10−4 mmol l−1 to 0.5 mmol l−1 range. The relation between the evolution of Ret and CHX-Dg concentration was found to be negatively correlated. When CHX-Dg was added, the electrochemical monitoring of the bacterial kinetic adhesion showed that the electrode's capacity (CP) variation remained stable, demonstrating that the addition of CHX-Dg in the broth inhibited bacterial adhesion.