Fixed-biomass reactors present the main advantage of increasing the hydrogen production rates by supporting high organic loading rates. However, the use of complex natural inoculum often precludes a long-term operation through the rapid emergence of methanogens. In this study, an Anaerobic Sequencing Batch Biofilm Reactor (ASBBR) was inoculated with a simplified H2-producing culture to evaluate its influence on robustness and more particularly on methanogenic activity. An ASBBR was operated over 6 months with unsterile lactose-rich effluent and under Hydraulic Retention Time ranging from 1 to 34 h. Hydrogen performances in terms of productivity and yields ranged from 0.23 to 519 mmoleH2 L−1 d−1 and 0.01 to 7.11 moleH2 molelactose−1, respectively. No significant methane production was observed all along the experimental procedure, showing that inoculating with a simplified and highly enriched preculture could increase substantially the robustness of the process. Specific preparation of the inoculum may represent a solution to sustain long-term operation of biofilm-based reactors.