Identification of microbes that actively utilize root exudates is essential to understand plant–microbe interactions. To identify active root exudate-utilizing microorganisms associated with switchgrass – a potential bioenergy crop – plants were labelled in situ with 13CO2, and 16S and 18S rRNA genes in the 13C-labelled rhizosphere DNA were pyrosequenced. Multi-pulse labelling for 5 days produced detectable 13C-DNA, which was well separated from unlabelled DNA. Methylibium from the order Burkholderiales were the most heavily labelled bacteria. Pythium, Auricularia and Galerina were the most heavily labelled eukaryotic microbes. We also identified a Glomus intraradices-like species; Glomus members are arbuscular mycorrhizal fungi that are able to colonize the switchgrass root. All of these heavily labelled microorganisms were also among the most abundant species in the rhizosphere. Species belonging to Methylibium and Pythium were the most heavily labelled and the most abundant bacteria and eukaryotes in the rhizosphere of switchgrass. Our results revealed that nearly all of the dominant rhizosphere bacterial and eukaryotic microbes were able to utilize root exudates. The enrichment of microbial species in the rhizosphere is selective and mostly due to root exudation, which functions as a nutrition source, promoting the growth of these microbes.