Large amounts of H2 are produced in the nodules of legume plants during N2-fixation, which often subsequently leaks to the surrounding soil environment. This leaked H2 activates H2-oxidizing bacterial (HOB) communities and thus can alter microbial community structure and soil carbon and nitrogen turnover; a process that may explain part of the positive effect of crop rotations with legumes. We tested this hypothesis in the field by infusing H2 in soil for 4 weeks and followed the effect on bacterial, archaeal and fungal communities by amplicon sequencing, as well as the abundance of genes involved in the nitrogen cycle using quantitative real-time PCR (qPCR). Soil exposure to elevated H2 levels changed the capacity of soil to scavenge H2 with a stimulation of low-affinity H2-oxidizing activity. Activation of this functional group of bacteria led to significant increases in the relative abundance of Nitrososphaera, Gaiella and Gp16 in soil bacterial and archaeal communities, albeit without changing their diversity. In contrast, H2 infusion had little effect on fungal community diversity, composition and taxa abundance. The abundance of selected functional genes encoding for bacterial and archaeal enzymes involved in the nitrogen cycle was not influenced by H2 infusion, but the abundance of nifH was significantly affected by H2 infusion × sampling date interaction. Variation in the high-affinity and low-affinity HOB, microbial community diversity and composition, and N-cycle gene abundance between sampling dates was attributed, in part, to significant differences in soil moisture during the study. Together, this work confirms that microbial taxa differ in their reaction to H2 exposure in the field, and provides the first evidence that nitrifier and denitrifier microorganisms have uneven responses to elevated H2 levels under field conditions.