The main purpose of this study was to identify the microbial communities (bacterial, archaeal and fungal) in a two-stage system of anaerobic bioreactors for the production of hydrogen and methane from the waste substrate—corn steep liquor. Wastes from the food industry are valuable resources with potential in biotechnological production because of their high organic matter contents. In addition, the production of hydrogen and methane, volatile fatty acids, reducing sugars and cellulose content was monitored. Two-stage anaerobic biodegradation processes were performed by microbial populations in the first hydrogen generating bioreactor (working volume of 3 dm3) and in the second methane-generating reactor (working volume of 15 dm3). Cumulative hydrogen yield reached 2000 cm3or 670 cm3/L a day, while the methane production reached a maximum quantity of 3300 cm3or 220 cm3/L a day. Microbial consortia in anaerobic digestion systems play an essential role for process optimization and biofuel production enhancement. The obtained results showed the possibility of conducting two separate processes—the hydrogenic (hydrolysis and acidogenesis) and methanogenic (acetogenesis and methanogenesis)—as two stages of anaerobic digestion to favor energy production under controlled conditions with corn steep liquor. The diversity of microorganisms as main participants in the processes in the bioreactors of the two-stage system was followed using metagenome sequencing and bioinformatics analysis. The obtained metagenomic data showed that the most abundant phylum in both bacterial communities wasFirmicutes—58.61% and 36.49% in bioreactors 1 and 2, respectively. PhylumActinobacteriawere found in significant quantities (22.91%) in the microbial community in Bioreactor 1, whereas in Bioreactor 2, they were 2.1%.Bacteroidetesare present in both bioreactors. PhylumEuryarchaeotamade up 0.4% of the contents in the first bioreactor and 11.4% in the second. As the dominant genera among methanogenic archaea areMethanothrix(8.03%) andMethanosarcina(3.39%), the main fungal representatives wereSaccharomyces cerevisiae. New knowledge of anaerobic digestion mediated by novel microbial consortia could be widely used to convert different wastes to green energy.