The impacts of ZVI on microbial community diversity in an anaerobic assay with high organic loading were investigated. The relative abundance of bacteria, archaea, and the functional methyl coenzyme-M reductase (mcrA) gene were investigated using high-throughput sequencing, and variations in their quantity were determined by qPCR. The results showed that ZVI significantly increased both the relative abundance and quantity of Methanobacteriales and Methanosarcinales during hydrogenotrophic and acetoclastic methanogenesis. The relative abundance of syntrophic Methanobacteriales at the hydrolysis and acidogenesis stages resulted in H-2 partial pressure decrease through an interspecies hydrogen transfer (IHT) network, which further induced butyric conversion to acetic by Syntrophomonas. The primary microbial metabolism then converted to acetoclastic methanogensis in the assay with ZVI addition. The short duration of this process and high relative abundance of Syntrophomonas, Clostridium butyricum and Methanosarcinales potentially indicated the existence of a novelty syntrophic mechanism for extracellular electron transfer, which promoted CH4 generation.