The search for sustainable development has led countries around the world to seek the improvement of technologies that use renewable energy sources. One of the alternatives in the production of renewable energy comes from the use of waste including urban solids, animal excrement from livestock and biomass residues from agro-industrial plants. These materials may be used in the production of biogas, making its production highly sustainable and environmentally friendly, in addition to reducing public expenses for the treatment of those wastes. The present study evaluated the cultivated and uncultivated microbial community from a substrate (starter) used as an adapter for biogas production in anaerobic digestion processes. 16S rDNA metabarcoding revealed domain of bacteria belonging to the phyla Firmicutes, Bacteroidota, Chloroflexi and Synergistota. The methanogenic group was represented by the phyla Halobacterota and Euryarchaeota. Through 16S rRNA sequencing analysis of isolates recovered from the starter culture, the genera Rhodococcus, Vagococcus, Lysinibacillus, Niallia, Priestia, Robertmurraya, Luteimonas and Proteiniclasticum were recovered, groups that were not observed in the metabarcoding data. The groups mentioned are involved in the metabolism pathways of sugars and other compounds derived from lignocellulosic material, as well as in anaerobic methane production processes. The results demonstrate that culture-dependent approaches, such as isolation and sequencing of isolates, as well as culture-independent studies, such as the Metabarcoding approach, are complementary methodologies that, when integrated, provide robust and comprehensive information about the microbial communities involved in various processes, including the production of biogas in anaerobic digestion processes.