Model hyperthermophilic microorganisms Pyrococcus furiosus and Thermotoga maritima were investigated as sources of thermostable and thermoactive enzymes for polysaccharide hydrolysis as well as for their capacity to produce biohydrogen from carbohydrate-based feedstocks. A functional genomics approach to biocatalyst discovery was used to identify ORFs encoding new glucan-hydrolyzing enzymes and to provide clues as to the physiological role of these glycoside hydrolases. Biohydrogen production by these hyperthermophiles was strongly affected by the carbohydrate used as primary carbon/energy source. For P. furiosus, glucan linkage (cellobiose vs. maltose) and the availability of elemental sulfur had a profound effect on bioenergetics, hydrogen generation rates, as well as the transcriptome. For T. maritima, co-fermentation of glucose and xylose was studied and compared to fermentation patterns for the individual sugars. Significant interaction between hexose and pentose processing pathways was found to impact hydrogen production and this observation could be interpreted in terms of transcriptional response analysis. Taken together, these results support the potential importance of hyperthermophilic microorganisms in biofuels production.