This study evaluates the biological water-gas shift reaction of syngas into hydrogen (H2) using natural microbial consortia from wastewater treatment facilities. Various inoculum pretreatment techniques to inhibit methanogenesis and different operational conditions such as temperature, pH, and inoculum concentrations were tested to improve H2 production. An inoculum heat pretreatment of 85 °C for 30 min followed by a fermentation temperature of 65 °C and an initial pH value of 9, resulted in the maximum CO-to-H2 conversion (2.35 ± 0.00 mmol) in which Firmicutes dominated with a total inhibition of methanogens, such as with the chemical inhibition treatment. Additionally, volatile fatty acids (VFA) production was observed, being influenced by the pre-treatment. This study highlights the potential of natural consortia for efficient, cost-effective and scalable syngas fermentation processes, offering new insights into the bioconversion of H2 without costly chemical inhibitors.