Sudden death syndrome (SDS) caused by Fusarium virguliforme is a widespread and economically important disease of soybean.SDSis typically distributed unevenly in patches across soybean fields. While certain spots in fields are highly conducive to the development of severe SDS, other areas appear to be naturally healthy or suppressive to the disease. The role of soil microbial communities and soil physical and chemical properties in SDS development was investigated in 45 soybean fields in Illinois, Iowa, and Minnesota. Soil samples were collected from symptomatic patches in fields and from adjacent areas where SDS foliar symptoms did not develop. Multiple edaphic factors were measured, and markers specific to bacteria, fungi, archaea, oomycete, and nematodes, coupled with Illumina MiSeq sequencing, were used to identify key taxa likely associated with SDS development. A total of 14,200,000 reads were mapped against the National Center for Biotechnology Information nucleotide database and taxonomically compared using Metagenome Analyzer (MEGAN5). Physical and chemical analysis of soil variables did not provide any correlation with the patchiness pattern of SDS. Significant differences in bacterial and fungal community structure between soils from healthy and diseased areas of fields were found. At least 20 taxa were associated with healthy soils, including members of Fusarium oxysporum sp. complex, Actinomycetales, Firmicutes, Chloroflexi, Trichoderma spp., Pseudomonas, Metacordyceps, Penicillium, Purpureocillium, and Myceliophtora. In diseased soils, Fusarium solani spp., Phallus rugulosus, Stachybotrys, and Alpha- and Betaproteobacteria dominated. Our results suggest that the relative abundance of multiple microbial taxa in the soil plays a key determinant in the incidence of SDS.