Information about the root mycobiome may improve the overall quality of the plants and contribute to a valuable strategy to enhance sustainable agriculture. Therefore, we assessed differences in fungal community diversity and composition in the roots of potato, wheat and barley grown under mineral nitrogen fertilisation at five rates, with and without farmyard manure amendment. The same factorial combination of treatments has been used since 1989. Species richness and diversity, as well as community composition, of different fungal guilds were characterised using Illumina MiSeq sequencing of the ITS2 region. Crop species was the main factor determining overall fungal richness and diversity, with wheat showing the highest, and potato the lowest, richness and diversity. Pathogen diversity indices were highest in wheat plots amended with farmyard manure, whereas the lowest values were observed for potato roots. Fertilisation treatments and the interaction between crop species and fertilisation had the strongest impact on arbuscular mycorrhiza and saprotroph diversity. Crop species also determined the composition of the overall fungal community and that of fungal guilds, whereas fertilisation treatment had only a minor effect. This study highlights crop species as the main driver in shaping root fungal diversity and composition under the same environmental conditions.