This study set out to cultivate and isolate hydrogen-oxidising bacteria (HOB) for microbial protein production under a specific culture strategy with a particular focus on assessing the influence of different environmental sources on enrichment culture and strain diversity. Therefore, HOB were enriched from samples collected from various freshwater lakes and streams, and novel strains were subsequently isolated from these cultures. The enrichment procedure revealed significant shifts in community compositions, which were mainly driven by changes in the relative abundance of genera affiliated to Pseudomonadota and Bacteroidota. Sample-specific variations were observed in the communities of the inocula, reflecting distinct community structures associated with distinct ecological functions. The most common autotrophic HOB, Hydrogenophaga, proliferated in some of the cultures. However, several genera, such as Acinetobacter and Klebsiella that have not been previously recognised with hydrogen-oxidation characteristics, were also enriched, suggesting potential novel contributors to HOB communities.