This study unravels some of the complex microbial interactions established in the fermentation of sugarcane vinasse. The microbial communities of four thermophilic (55 °C) fixed-bed vinasse-fed reactors were analyzed by 16S rRNA gene amplicon sequencing and the marker gene data were aligned with reference genomes to predict metabolic functions of the microorganisms. The production (by Lactobacillus) and utilization (mainly by Thermoanaerobacterium and Caproiciproducens) of lactate explain the primary pathways associated with substrate fermentation towards biohydrogen, whilst only a single genus (Desulfotomaculum) and very low abundances of the enzymes involved in sulfate reduction were found. Although the abundant microbes in the inocula failed to grow during the continuous operation in all reactors, these fermentative consortia behave like microbial pantries containing bacteria that will grow according to the operating conditions, shaping lactate-accumulating (pH < 5.0) or lactate-consuming (pH > 5.0) and eventually sulfate-reducing (pH > 6.0) microbial communities in vinasse thermophilic fermentation.