With its high energy content and clean combustion, hydrogen is recognized as a renewable clean energy source with enormous potential. Biological hydrogen production is a promising alternative with significant advantages over conventional petroleum-derived chemical processes. Sustainable hydrogen production from renewable resources such as cassava, wastewater, and other agricultural waste is economically feasible for industrial applications. So far, the major bottlenecks in large-scale biological hydrogen production are the low production rate and yield. This review discusses the various factors that affect the metabolic pathways of dark hydrogen production, and highlights the state-of-the-art development of mixed culture technology. The aim of this review is to provide suggestions for the future directions of mixed culture technology, as well as by-product valorization in dark fermentation. This article is protected by copyright. All rights reserved.