Our research determined the impact of operational conditions on the quantity of total and ammonia-oxidizing bacteria (AOB) in immobilized biomass. The experiment was conducted for two kinds of wastewater differing in organic carbon concentration at HRTs of 1.5 h, 1.0 h, and 0.5 h. The evaluation of bacteria number was accomplished by using the real-time PCR targeting ammonia monooxygenase (amoA) and 16S rDNA genes. In the absence of organic carbon in the influent, the shortening of HRT from 1.5 h to 0.5 h positively affected the AOB number in immobilized biomass, which reached even 30%. But their activity was limited. In the presence of organic carbon in the influent, the shortening of HRT below 1.5 h resulted in a decline of the abundance of ammonia-oxidizing bacteria and nitrification efficiency.