Syngas fermentation offers a promising route for biological hydrogen (H2) production, yet industrial application is restrained by low yields and limited understanding of nutrient requirements in natural microbial consortia. This study investigated the macro- and micronutrient roles influencing carbon monoxide (CO)-to-H2 conversion in a thermophilic consortium using a medium for thermophilic, anaerobic, and carboxydotrophic microorganisms as the starting formulation. Yeast extract and trace element solution showed the strongest positive associations with H2 production, supplying essential vitamins, nitrogen, and enzymatic cofactors, including Ni and Fe, for CO dehydrogenase and hydrogenase activity. In contrast, sulfide solution and NH4Cl showed inhibitory trends, likely through redox imbalance and hydrogenase suppression. Optimizing the concentrations of yeast extract (7.5 g/L) and trace element solution (4 mL/L) increased H2 production by 53% (3.59 ± 0.06 mmol), although the elevated volatile fatty acid concentration indicated partial reliance on organic carbon metabolism. Reducing yeast extract to 0.5 g/L and supplementing with Wolin’s vitamin solution maintained equivalent productivity while reducing medium costs by approximately 45% relative to DSMZ 507. These findings suggest that precise nutrient management plays a decisive role in governing CO conversion toward H2, providing critical insights for scalable, economically viable biohydrogen production from syngas fermentation.