Basidiomycota constitute a group of fungi with highly diverse ecological strategies from ectomycorrhizas and plant pathogens to saprotrophic fungi. We used 454 sequencing to obtain sequences of Basidiomycota from a cultivated and an adjacent fallow field, to investigate the influence of soil cultivation on the species composition and distribution patterns. Nonparametric multidimensional scaling (NMS) clearly segregated the basidiomycetes communities in the two fields, though some species were shared. Axis 1 and 2 of the ordination correlated with soil organic matter content and pH respectively. Most OTUs had affinity to known species, but some unknown clades were seen in both fields. Spatial analysis demonstrated higher spatial autocorrelation (up to 50 m) in the managed field in comparison with the fallow field. The study showed that agricultural soils have considerable species richness, and that community composition and turn-over potentially is influenced both by land use and spatial scale.