Tertiary wastewater treatment could provide a reliable source of water for reuse. Amongst these types of wastewater treatment, deep-bed filtration of secondary effluents can effectively remove particles and organic matter; however, NH₄⁺ and NO₂⁻ are not easily removed. This study examined the feasibility of stimulating microbial activity using hydrogen peroxide (H₂O₂) as a bio-specific and clean oxygen source that leaves no residuals in the water and is advantageous upon aeration due to the solubility limitations of the oxygen. The performance of a pilot bio-filtration system at a filtration velocity of 5–6 m/h, was enhanced by the addition of H₂O₂ for particle, organic matter, NH₄⁺, and NO₂⁻ removal. Hydrogen peroxide provided the oxygen demand for full nitrification. As a result, influent concentrations of 4.2 ± 2.5 mg/L N-NH₄⁺ and 0.65 ± 0.4 mg/L N-NO₂ were removed during the short hydraulic residence time (HRT). In comparison, filtration without H₂O₂ addition only removed up to 0.6 mg/L N-NH₄⁺ and almost no N-NO₂⁻. A DNA metagenome analysis of the functional genes of the media biomass reflected a significant potential for simultaneous nitrification and denitrification activity. It is hypothesized that the low biodegradability of the organic carbon and H₂O₂ addition stimulated oxygen utilization in favor of nitrification, followed by the enhancement of anoxic activity.