The fouling of periphyton biofilm is a common problem associated with surface water treatment systems. In this study, sulfuric acid (H2SO4), sodium acetate (CH3COONa) and ethylenediamine tetra-acetic acid (EDTA) solutions were sequentially used to clean periphyton biofilms collected from a surface water treatment system. The results showed that the sequential addition of H2SO4 and CH3COONa solutions could accelerate the exfoliation of the fouled periphyton biofilm, while the addition of EDTA solution could regenerate the periphyton biofilm. However, the addition of H2SO4 and CH3COONa solution might negatively affect the bacterial community structure, while the addition of EDTA solution facilitated improvement of the community structure. The combined effect of cleaning and regeneration of periphyton biofilm has significantly improved the removal efficiencies of chemical oxygen demand (COD), total phosphorus (TP), total nitrogen (TN) and ammonia (NH4-N), by 19, 20, 23 and 22%, respectively. The removal processes of COD, TP, TN and NH4-N by the cleaned biofilm were fitted to power regression curves, while those by the control biofilm during the removal process were fitted to polynomial regression curves. These systemic results indicate that the sequential addition of H2SO4, CH3COONa and EDTA solution is able to clean fouled periphyton biofilm and to enhance the efficiency of surface water treatment systems.