Peucedanum alsaticum and Peucedanum cervaria represent characteristic umbellifers (Apiaceae) of calcareous grasslands in Central and Eastern Europe. Both accumulate glucose, fructose, mannitol and sucrose as dominant carbohydrates in their roots. The objective of the study was to determine if endophytes utilise host plant carbohydrates differently than rhizosphere and bulk soil microfungi. Inula ensifolia (Asteraceae), Lathyrus latifolius (Fabaceae) and Bromus erectus (Poaceae), all plants that grow along with the two umbellifers, accumulated only sucrose as major sugar in their roots. Germ tube growth of 30 microfungal isolates, recovered from various rhizosphere habitats, was quantified in microdilutions (10–5000 μg ml−1) of a number of substrates, including glucose, sucrose, mannitol and the water-soluble plant root carbohydrate mixtures. Multivariate analysis of variance and subsequent least significant difference analysis of isolate group means revealed that sucrose and mannitol utilisation affected affiliation to a certain fungal lifestyle. Endophytes utilised host plant carbohydrates more efficiently at lower concentrations. Conversely, higher concentrations slowed their growth. Non-host carbohydrates did not cause comparable effects. The results suggest that root carbohydrate diversity may determine fungal diversity in natural rhizosphere environments.