Hydrogen-yielding fermentation conducted in bioreactors is an alternative method of hydrogen production. However, unfavourable processes can seriously inhibit bio-hydrogen generation during the acidogenic step of anaerobic digestion. Here, ascomycetous yeasts were identified as a major factor inhibiting the production of bio-hydrogen by fermentation. Changes in the performance of hydrogen-producing bioreactors including metabolic shift, quantitative changes in the fermentation products, decreased pH, instability of the microbial community and consequently a dramatic drop in bio-hydrogen yield were observed following yeast infection. Ascomycetous yeasts from the genera Candida, Kazachstania and Geotrichum were isolated from hydrogen-producing bioreactors. Yeast metabolites secreted into the growth medium showed antibacterial activity. Our studies indicate that yeast infection of hydrogen -producing microbial communities is one of the serious obstacles to use dark fermentation as an alternative method of bio-hydrogen production. It also explains why studies on hydrogen fermentation are still limited to the laboratory or pilot-scale systems. (C) 2018 The Author(s). Published by Elsevier Ltd on behalf of Hydrogen Energy Publications LLC.