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The back-diffusion of inactive gases severely inhibits the hydrogen (H2) delivery rate of the close-end operated hydrogen-based membrane biofilm reactor (H2-based MBfR). Nevertheless, less is known about the response of microbial communities in H2-based MBfR to the impact of the gases’ back-diffusion. In this research, the denitrification performance and microbial dynamics were studied in a H2-based MBfR operated at close-end mode with a fixed H2 pressure of 0.04 MPa and fed with nitrate (NO3−) containing influent. Results of single-factor and microsensor measurement experiments indicate that the H2 availability was the decisive factor that limits NO3− removal at the influent NO3− concentration of 30 mg N/L. High-throughput sequencing results revealed that (1) the increase of NO3− loading from 10 to 20–30 mg N/L resulted in the shift of dominant functional bacteria from Dechloromonas to Hydrogenophaga in the biofilm; (2) excessive NO3− loading led to the declined relative abundance of Hydrogenophaga and basic metabolic pathways as well as counts of most denitrifying enzyme genes; and (3) in most cases, the decreased quantity of N metabolism-related functional bacteria and genes with increasing distance from the H2 supply end corroborates that the microbial community structure in H2-based MBfR was significantly impacted by the gases’ back-diffusion.