In biological nitrogen removal, application of the autotrophic anammox process is gaining ground worldwide. Although this field has been widely researched in last years, some aspects as the accelerating effect of putative intermediates (mainly N₂H₄ and NH₂OH) need more specific investigation. In the current study, experiments in a moving bed biofilm reactor (MBBR) and batch tests were performed to evaluate the optimum concentrations of anammox process intermediates that accelerate the autotrophic nitrogen removal and mitigate a decrease in the anammox bacteria activity using anammox (anaerobic ammonium oxidation) biomass enriched on ring-shaped biofilm carriers. Anammox biomass was previously grown on blank biofilm carriers for 450 days at moderate temperature 26.0 (±0.5) °C by using sludge reject water as seeding material. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. With addition of 1.27 and 1.31 mg N L⁻¹ of each NH₂OH and N₂H₄, respectively, into the MBBR total nitrogen (TN) removal efficiency was rapidly restored after inhibitions by NO₂⁻. Various combinations of N₂H₄, NH₂OH, NH₄⁺, and NO₂⁻ were used as batch substrates. The highest total nitrogen (TN) removal rate with the optimum N₂H₄ concentration (4.38 mg N L⁻¹) present in these batches was 5.43 mg N g⁻¹ TSS h⁻¹, whereas equimolar concentrations of N₂H₄ and NH₂OH added together showed lower TN removal rates. Intermediates could be applied in practice to contribute to the recovery of inhibition-damaged wastewater treatment facilities using anammox technology.