A lab-scale partial nitrifying sequencing batch biofilm reactor was a successful start-up. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial community dynamics in three periods together with inocula sludge at ambient temperature. The DGGE profiles of bacteria and Shannon-Wiener index (H') results showed that high free ammonia (FA) concentration referred to lower diversity in the bioreactor system. Cluster analysis indicated that microorganism in period III was similar with inocula sludge and was different from that in periods I and II. Similar results also appeared in ammonia-oxidizing bacteria (AOB) community structure and nitrite-oxidizing bacteria (NOB) community structure, and at least four AOB species and two NOB species were present in period III, respectively. Phylogenetic analysis of amoA gene sequences showed that Nitrosomonas eutropha cluster was predominant in all the three periods. With lower ammonium loads, three new operational taxonomic units formed and consisted Nitrosomonas sp. Cluster. This article demonstrated that microbial community, AOB, and NOB diversity were related with FA concentration closely at ambient temperature.