Producers, consumers and decomposers are the three key functional groups that form the basis of all ecosystems. But, little is known about how these functional groups coexist with each other in aquatic environment, particularly in subtropical reservoirs. In this study, we describe the nature of microeukaryotic communities in a subtropical deep reservoir during the strongly stratified period. Denaturing gradient gel electrophoresis gel band sequencing, pyrosequencing and light microscopy were used together to facilitate an in-depth investigation of the community structure of phytoplankton, zooplankton and fungi. Our results showed that thermal and oxygen stratification shaped the composition of the phytoplankton, zooplankton and fungi populations in the reservoir. Stratification was evident among ecological functional groups in autumn: producers and consumers were overwhelmingly dominant in the epilimnion characterized by high temperatures and oxygen levels, while decomposers were inclined to inhabit the hypolimnion. These results contribute to our understanding of the relationship of ecosystem functional groups in the man-made aquatic systems and have important practical implications for reservoir management. Results suggest that the strategies for the control of eutrophication and harmful algal bloom prevention should focus on a fuller understanding of the consequences of both thermal stratification and vertical distribution of microplankton.