The aim of this study is to evaluate the effect of different thermal pretreatments of the inoculum on the diversity of the microbial community producing hydrogen from sugarcane vinasse. High-throughput sequencing of the 16S and 18S rRNA genes was performed. The reactor samples were also selected for the isolation of strict anaerobes. Decreased microbial diversity was observed with increasing pretreatment temperatures, with Firmicutes predominating: 90% to 97%. The highest abundance of Staphylococcus (7.9%) was found in pretreatment at 120 °C / 20 min at pH 6. The fungal analysis revealed a high prevalence of Candida (47%), Agaricomycetes, Pezizomycotina and Aspergillus in assays with higher H2 production (90° C / 10 min at pH 6). Three species of Clostridium were isolated: C. bifermentans, C. saccharoperbutylacetonicum and C. saccharobutylicum. The isolates were tested separately and in co-cultures for the production of hydrogen. Hydrogen-producing capacity by co-culture of Clostridium species was increased by 18%. Knowing microorganisms and understanding the interaction between eukaryotes and prokaryotes is essential to obtain strategies for biotransformation of vinasse for the production of bioenergy.