We have previously identified Caldicellulosiruptor spp. as potential candidates for biohydrogen production since they efficiently produce hydrogen from various carbohydrates at high yields. In the present work, we combined two Caldicellulosiruptor spp. in a defined co-culture for biohydrogen production. The population dynamics in a continuous hydrogen-production system under different conditions were followed using quantitative real-time PCR. Interestingly, the two species stably co-existed in the system under both carbon and non-carbon limited conditions. The hydrogen yields obtained by the co-culture at higher residence times were close to the theoretical maximum, as acetate was the main metabolic end product detected. These yields are at least twice as high as those obtained by most previously reported mixed cultures. Carbon limitation improved the sugar conversion efficiency and slightly increased hydrogen yield and productivity. Depending on the substrate, the developed co-culture might offer an attractive alternative to the traditional “undefined” mixed culture for a cost-effective biohydrogen process.