Despite the advantages of the next generation sequencing (NGS) techniques, one of their caveats is that they do not differentiate between microbes that are actively participating in carbon cycling in the rhizosphere and microbes performing other functions in the soils. Here we combined DNA-SIP with NGS to investigate which rhizosphere fungi actively assimilate plant-derived carbon. We provided 13CO2 to plants in intact soil cores collected from a grassland and sampled the rhizosphere in a time series to follow the fate of carbon in the rhizosphere mycobiome. We detected a difference between active rhizosphere fungi using plant-derived carbon and the total mycobiota: 58% of fungal species were using fresh rhizodeposits, and an additional 22% of fungal species received carbon several weeks later while 20% were not involved in cycling of freshly photosynthesized carbon. We show that members of Ascomycota, Mucoromycota, and basidiomycete yeasts were first users of freshly photosynthesized carbon, while fungi not using recently fixed carbon consisted mainly of mycelial (non-yeast) Basidiomycota. We conclude that a majority of fungi inhabiting the rhizosphere in this grassland ecosystem are actively using plant derived carbon either directly or via food-web interactions.