To investigate the effects of different substrates on the biodiversity and hydrogen production performance of microbial electrolysis cell (MEC) anodic membranes, the vital electroactive microorganisms (e.g. in MEC hydrogen production) were worth identifying. In the present study, single-factor experiments were performed. Sodium acetate, sodium propionate, sodium butyrate, glucose and starch served as different substrates for MEC anodic culture experiments under the same condition. The effects of different substrates on the bioactivity, biomass and hydrogen production performance of MEC anodic films were analyzed. Also, the effects of different microbial communities on hydrogen production were studied using 16S rRNA sequencing. According to the experimental results, all the five substrates here can serve as hydrogen-producing raw materials for MEC. All indicators revealed that sodium acetate, sodium propionate and sodium butyrate are excellent biofilm culture materials. The serious acidification of glucose and starch was identified at the same substrate concentration, and the environment of the culture medium was difficult to control, which affected the growth and metabolism of electroactive microorganisms. In comparison, sodium acetate was the best, achieving a maximum output of 23.4 mA and a maximum hydrogen content of 25.85%. The other four were ranked as sodium butyrate > sodium propionate > glucose > starch. According to the results of high-throughput sequencing, when sodium acetate, sodium propionate, sodium butyrate, glucose and starch served as substrates, the number of operational taxonomic units reached 464, 728, 636, 784, and 1,083, respectively. Furthermore, when MEC was cultured with sodium acetate, sodium propionate and sodium butyrate as substrates, the electroactive microorganism Desulfuromonas in the Proteobacteria would contribute the most to producing hydrogen. The relative abundance of the five substrates was ranked as sodium acetate > sodium butyrate > sodium propionate > glucose > starch, suggesting that the MEC anodic film cultured with sodium acetate as the substrate exhibited the best hydrogen production performance, and the starch showed the worst. It is noteworthy that Desulfuromonas was the most abundant species according to sequencing results. When glucose and starch served as substrates, they exhibited high biodiversity. The anodic membranes cultured with sodium acetate, sodium propionate and sodium butyrate were not as good as those cultured with glucose and starch, yet the electroactive microorganisms were up-regulated.