Aims: To obtain insight into the complex behaviour of denitrifying and total bacterial groups during the nitrogen accumulation process in an ammonia-loaded biofiltration system. Methods and Results: Denitrifying and total bacterial communities in a laboratory-scale rockwool biofilter with intermittent water recirculation were analysed by using denaturing gradient gel electrophoresis targeting nosZ and metabarcoding sequencing of the 16S rRNA gene. Gene abundance was evaluated by quantitative PCR. The nosZ number increased from 6.59 x 10(6) to 3.33 x 10(8) copies per gram dry sample over the 436 days of operation, during which nitrogen mass balance errors increased to 39%. The nosZ sequences associated with the genera Castellaniella, Hyphomicrobium and Pseudomonas were detected. Metabarcoding sequencing analysis indicated that the proportions of the genera for which at least one denitrifying strain or species possessing nosZ had been characterized corresponded well to the nitrogen loss. In addition, the genus Nitrosococcus (gamma-proteobacteria) increased its relative abundance at days 317 and 436. Conclusions: The increased proportion of denitrifying bacteria in this ammonia-loaded biofiltration system could be related to the nitrogen loss. Significance and Impact of the Study: These results will help to clarify the complex behaviour of nitrifiers and denitrifiers within ammonia-loaded biofiltration systems.