The objective of this study was to identify the catabolic pathways and genes utilized by Clostridium thermocellum ATCC 27405 during both hydrogen (H2) and ethanol synthesis. Bioinformatic analyses of the C. thermocellum genome identified genes encoding key enzymes in pyruvate catabolism pathways, including two putative lactate dehydrogenases, one pyruvate:formate lyase, four pyruvate:formate lyase activating enzymes, and at least three pyruvate:ferredoxin oxidoreductase (POR) or POR-like enzymes. Quantitative polymerase chain reaction was used to confirm the expression of the identified genes throughout growth on α-cellulose. Our data suggests H2 may be generated through the action of either a ferredoxin (Fd)-dependent, membrane-bound NiFe hydrogenase, often referred to as “Energy-converting Hydrogenases”, or via NAD(P)H-dependent Fe-only hydrogenases. Furthermore, our findings show the presence of a gene cluster putatively encoding NADH:Fd oxidoreductase; suggesting a mechanism in which electrons may be transferred between NADH and ferredoxin. Ethanol production appears in part to be catalyzed by an ADH-E enzyme possessing both acetaldehyde and alcohol dehydrogenase activities. The results presented here provide insight into the metabolic pathways utilized in biofuels production. This information is critical to developing strategies for metabolic engineering and optimizing bioprocesses promoting enhanced and H2 gas or ethanol production.