Efficient gas delivery and biofilm development on membrane fibers in a membrane biofilm reactor (MBfR) would be well suited to autotrophic nitrification and denitrification using hydrogen. Total nitrogen removal in a two-step MBfR system incorporating sequential nitrification and hydrogen-driven autotrophic denitrification was investigated in order to achieve nitrogen removal by autotrophic bacteria alone. This study also aimed at the long-term stable operation, which proved difficult in previous studies due to excessive biofilm accumulation in autotrophic denitrification systems. Consecutive operation of nitrification and autotrophic denitrification lasted 230 days. Average specific nitrification rate of 1.87 g N/m2 d was achieved and the performance was very stable throughout the experimental periods. Nitrification performance from this study showed comparable rates to previous studies although this work was conducted at slightly lower temperature. Batch tests confirmed the presence of nitrifiers from the effluent of the nitrification reactor, which reattached to the biofilm in the denitrification reactor leading to further nitrification. Performance of autotrophic denitrification was maintained stably throughout the experimental periods, however biofilm control by nitrogen sparging was required for process stability. Average specific denitrification rate of 1.41 g N/m2 d and a maximum specific denitrification rate of 2.04 g N/m2 d was maintained. This study showed that, with an appropriate biofilm control plan, stable long-term operation of a fully autotrophic MBfR system for total nitrogen removal was possible without major membrane cleaning procedures.