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The efficacy of a fine bubble (FB) diffuser in enhancing the performance of an attached biofilm hydrogenotrophic denitrification (HD) reactor was evaluated. HD reactors equipped with an FB diffuser (FB reactor) and an air stone (AS) diffuser that produced ordinary bubbles (AS reactor) were operated in parallel at different hydraulic retention times (HRTs) in a synthetic groundwater treatment experiment. A reduction in H2 consumption of approximately 77% was achieved using the FB diffuser to reach a gas-liquid mass transfer coefficient similar to that of the AS diffuser. The high gas dissolution efficiency of the FB diffuser resulted in an effective nitrogen removal rate (NRR) enhancement, requiring less H2 supply. The highest value of NRR at 53.0±9.8 g-N/m3/d was obtained in the FB reactor at a minimum HRT of 3 h, which was two-fold higher than the corresponding value from the AS reactor. The FB reactor also had the lowest requirement of H2 for denitrification reaching 0.1 m3-H2/g-N in this condition, which was ten-fold lower than that in the AS reactor. Furthermore, the suspended sludge concentration in the FB reactor was lower than that in the AS reactor, indicating that the application of the FB diffuser can minimize excess suspended sludge accumulation inside the HD reactor. Microbial community analysis showed the predominance of Thauera spp. reaching a relative abundance of 15.7–27.3% in the FB reactor, suggesting a contribution to the HD. This finding can provide insight into the application of the FB diffuser for optimizing nitrate-contaminated groundwater treatment technology by HD.