A descriptive analysis of microbial communities from two different sediment locations in a subtropical reservoir was undertaken, using DGGE and 16S rRNA gene clone library. This study evaluated bacterial diversity in the sediment of a subtropical reservoir, based on quantitative and qualitative analyses of sulphate‐reducing bacteria (SRB), denitrifying bacteria (DB) and hydrogen‐producing bacteria (HPB). The sediment samples were taken from a subtropical reservoir (Ibiúna, SP, Brazil), being collected at two contrasting sites, including (A) at a 4 m depth and (B) at an 11 m depth of the water column. The anaerobic bacteria population density in the sediment was estimated with the most probable number (MPN) method. The microbial diversity of the sediment was assessed by molecular biology analyses for the domain Bacteria. Increased cell count numbers were observed in sample A for DB. The count for sample B was higher for SRB and HPB. Both sediment communities (A and B samples) exhibited a high diversity. They were similar to the phyla Proteobacteria, Chloroflexi, Firmicutes, Actinobacteria, Cyanobacteria, Fusobacteria, Deferribacteres, Verrucomicrobia and uncultivated bacteria. The environmental conditions of sediments from tropical climates were mainly sunlight during the day and temperatures around 25 °C, which would favour the maintenance of nutrients and the available organic matter for establishing the anaerobic bacteria populations. The microbial diversity observed for both samples indicates new possibilities for application of the sediment reservoir in regard to biotechnological processes such as the operation of anaerobic reactors for wastewater and toxic compound treatment, bioenergy generation and production of biogases such as hydrogen and methane.