Free ammonia inhibition to Nitrobacter population in a nitrifying biofilm was investigated. It was found that when the free ammonia concentration is greater than 0.1mgN/1, the oxidative activity of Nitrobacter was significantly inhibited, and resulting in a transient accumulation of nitrite ions. The results show that Nitrobacter population is capable of rapidly recovering its lost metabolic activity once the free ammonia concentration becomes less than 0.1mgN/1, and a complete oxidation of nitrite to nitrate was achieved.