Isoprene is a ubiquitous biogenic volatile organic compound (VOC) with atmospheric concentrations comparable to those of methane. Its high reactivity in the atmosphere significantly influences methane concentrations, contributing to adverse effects on the climate. Understanding the role of isoprene and its link to methane metabolism is crucial to addressing climate change. The fate of isoprene and its potential microbial degraders in soil, particularly in anaerobic environments, remains poorly investigated, underscoring the need for comprehensive studies. Our study provides physiological evidence for anaerobic microbial isoprene reduction and its influence on methanogenesis in Eucalyptus-leaf sediment. In our anaerobic culture-based studies, we have demonstrated that isoprene is reduced to three products, 3-methyl-1-butene, 2-methyl-1-butene, and 2-methyl-2-butene. Our findings suggest that Pelotomaculum sp. is capable of anaerobic isoprene reduction, as indicated by 16S rRNA analysis and dilution-to-extinction studies. Anaerobic microbial isoprene reduction simultaneously inhibited methane formation. Methanogenesis was completely inhibited in microcosm cultures amended solely with isoprene. In cultures supplemented with isoprene and carbon sources, only hydrogenotrophic methanogenesis was inhibited, indicating competition for hydrogen between the methanogens and isoprene degraders. Using a culture-based approach and molecular analysis, this study provides novel insights into the microbial dynamics of anaerobic isoprene reduction and the interplay between isoprene reducers and methanogens.