A lab-scale pure moving bed sequencing batch biofilm reactor (MBSBBR) was employed to investigate changes in nitrification kinetics and microbial diversity. The MBSBBR operated under different aeration strategies (defined by the ratio of the duration of the subphases with (t1) and without (t2) aeration (R = t2/t1)) − continuous (R = 0) and intermittent (with constant time of non-aerated subphases (t2 = 10 min) and variable duration of subphases with aeration (t1 = 40 min–R = 1/4, t1 = 30 min–R = 1/3, t1 = 20 min–R = 1/2) and dissolved oxygen (DO) concentrations (6 mg/L; 3.5 mg/L). Moreover, the reactor’s organic (OLR) and nitrogen (NLR) loading rates were changed in the following ranges: OLR—537–402 gCOD/m3·d, NLR—64–48 gN/m3·d. The obtained results showed that, irrespective of changes introduced in particular series, a highly effective nitrification process (93.36 ± 2.13%) was achieved. The activity of bacteria capable of oxidizing ammonia nitrogen changed differently from that of bacteria capable of oxidizing nitrites (NOB). An increase in R was the primary factor changing the activity of ammonia-oxidizing microorganisms. NOB activity was affected only by the reduction of OLR and NLR. NOB were the predominant bacterial group, consistent with the kinetics studies. A DO decrease caused an increase in the abundance of AOB, NOB, and Comammox bacteria. Comammox bacteria were the most abundant at R = 1/2 and DO = 3.5 mg/L.