Although drought and drying of waters occur globally, the effect of drying on sediment microbial communities underpinning aquatic biogeochemical processes is poorly understood. We used the molecular method of terminal-restriction fragment length polymorphism (T-RFLP) to assess changes in the microbial community structure of sediments undergoing different levels of inundation and drying within a reservoir during drawdown in a drought. Sediments with three hydrological conditions were investigated: dry sediments (no overlying water), littoral sediments (covered with 1–2 mm water) and inundated sediments (covered with >1 m water). Sampling was done in winter 2006 (August) and summer 2007 (January) in Lake Hume, Australia. The microbial communities differed significantly between the different levels of inundation at each sampling time. Community structure also changed significantly within each site between winter 2006 and summer 2007, possibly influenced by the change of season or protracted drying. Sites that were ‘littoral’ in winter 2006 became ‘dry’ in summer 2007, and became more similar to communities that were ‘dry’ at both sampling times. This suggested that the hydrological history of specific sites did not heavily influence the response of microbial communities to severe drying, and all communities undergoing ‘dry’ conditions within the summer 2007 sampling responded similarly.