The dynamic transition of bacterial community structure in a biofilm was monitored by the fluorescence in situ hybridization (FISH) technique and subsequent image analysis. Heterotrophic bacteria that had occupied the outer layer were gradually decreased whereas ammonia-oxidizing bacteria (AOB) gradually increased their growth activity and extended their existence area to the outer layer of the biofilm through the gradual reduction of the C/N ratio. The spatial organization of AOB in the biofilm dynamically changed responding to the environmental conditions such as pH fluctuation and lack of dissolved oxygen (DO) and had great influence on the nitrification activity. The accumulation of nitrite was observed at lower DO concentration, which might be due to the property that nitrite-oxidizing bacteria (NOB) possess of higher Km values for oxygen than AOB.