Recent studies have shown that free nitrous acid (FNA) is biocidal to a broad range of microorganisms. Microorganisms residing in anaerobic sewer biofilms were found to be inactivated after a short (6-24 h) exposure to FNA. In this study, we investigate the inactivation kinetics of anaerobic sewer biofilms grown in real wastewater. Microbial viability of biofilms was determined using LIVE/DEAD staining. A two-fraction kinetic model was developed to simulate the inactivation of mixed culture in biofilms. The kinetic parameters were estimated by using Bayesian statistics. Model simulation found that a fraction (85 %) of the biofilm community was highly sensitive to FNA with a high inactivation rate, and a fraction (15 %) was tolerant to FNA and persisted after FNA treatment. This different susceptibility to FNA treatment was likely due to the diverse microbial community and biofilm protection. The fact that nearly 85 % microbes were inactivated confirmed that FNA is a strong biocide to mixed-culture biofilms. It was found that the inactivation rate constant was not affected by pH levels. The kinetic model was successfully used to optimize FNA dosage for sulfide control in sewer biofilms. Also, results suggest that a high FNA concentration is preferred than long exposure time to reduce the total chemical consumption. © 2013 Springer-Verlag Berlin Heidelberg.