To the best of our knowledge, there was little information available on pathogen removal using low level disinfectant followed by free chlorine in sequential disinfection (SD). This study investigated Escherichia coli inactivation by four types of disinfection: single step disinfection (SSD), SD, traditional sequential disinfection (TSD) and mixed disinfectant disinfection (MDD). Results indicated that SD had higher ability to inactivate E. coli than the others, indicating there was a positive synergistic effect on chlorine disinfection by prior dosing with a low level of chlorine dioxide (ClO(2)). The ONPG assay suggested that the permeability of cell wall rather than the viability of E. coli were changed under 0.02 mg/l ClO(2) treatment. The coexistence of residual ClO(2) and free chlorine also plays an active synergistic effect. Additionally, temperature had a positive effect on E. coli inactivation in SD, while inactivation was reduced in alkaline compared to neutral and acidic conditions.