Microbial chain elongation (CE) has been shown at laboratory scale to drive reductive dehalogenation (RD) of chlorinated ethenes through both primary (oxidation of ethanol) and secondary (fermentation of medium chain carboxylates) hydrogen (H2) production. This process can offer engineers a sustainable in situ bioremediation alternative to address the challenges of conventional treatment technologies and processes. To aid in moving this process into field scale applications, a greater understanding of the specific microbiomes involved in both primary and secondary processes is needed. In this study, microbial community analysis was conducted on groundwater microcosms under various CE substrate combinations to quantify the extent of CE and the effect on RD of cis-1,2-dichloroethene (cis-DCE). Taxonomic classification of amplicon sequence variants obtained from DNA extracted from groundwater microcosms were used to characterize microbiomes using QIIME 2. Pielou’s eveness and beta diversity (via unweighted UniFrac distances) analyses were performed to assess the diversity of microbiomes. Overall, low concentration microcosms (excluding L-7:1 EtOH:Butyrate and L-9:1 EtOH:Acetate + Soil) underwent complete RD, as evidenced by significant ethene production. Alpha and beta diversity analyses confirm the findings of chemical data that the overall substrate concentrations played a major role in determining the extent of CE and RD.