Free nitrous acid (FNA) was recently revealed to be a strong biocide for microbes in anaerobic biofilm, achieving approximately 1-log (90%) inactivation at a concentration of 0.2-0.3mgHNO2-N/L with an exposure time longer than 6h. The combined biocidal effects of FNA and hydrogen peroxide (H2O2) on anaerobic wastewater biofilm are investigated in this study. H2O2 greatly enhances the inactivation of microorganisms by FNA. About 2-log (99%) of microbial inactivation was achieved when biofilms were exposed to FNA at 0.2mgN/L or above and H2O2 at 30mg/L or above for 6h or longer. It was found, through response surface methodology and ridge analysis, that FNA is the primary inactivation agent and H2O2 enhances its efficiency. The loss and the subsequent slow recovery of biological activity in biofilm reactors subjected to FNA and H2O2 dosing confirmed that the chemical combination could achieve higher microbial inactivation than with FNA alone. Reaction simulation shows that intermediates of reactions between FNA and H2O2, like peroxynitrite and nitrogen dioxide, would be produced at elevated levels and are likely responsible for the synergism between FNA and H2O2. The combination of FNA and H2O2 could potentially provide an effective solution to sewer biofilm control. © 2013 Elsevier B.V.