Fungal communities in soil have significant influences on terrestrial ecosystem dynamics, yet our understanding of the drivers of fungal diversity and community structure in soil is limited. Fungal communities associated with the rhizosphere of four native perennial grassland plant species, two legumes and two grasses, grown in monoculture and polyculture in a long-term field experiment were characterized. Reference databases were developed for, and amplicon libraries sequenced from, multiple-copy rRNA and single-copy protein-coding loci. Clustering and alignment-based pipelines were utilized to evaluate differences in fungal community structure and diversity in response to plant host, plant community richness, and soil edaphics. Fungal diversity increased in the rhizosphere of plants growing in polyculture plant communities as compared to monoculture plant communities. Fungal community structure was differentiated between legumes and grasses growing in monoculture but not in polyculture. To specifically monitor fungi in the genus Fusarium in the soil, the protein-coding locus was used to increase phylogenetic resolution and enrich for this taxon. These data show that fungal community richness and structure are strongly linked with plant community dynamics and associated soil edaphic characteristics in these grassland soils.