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Rotation is an important cultural practice that farmers use to manage crop growth and diseases. Diversified crop rotations directly affect plant residue inputs that may enrich specific groups of microbes that form beneficial associations with the following crop. At two field sites, yield differences were observed in soybeans planted after the following four long-term (14 year) crop sequences: continuous corn (CC), corn (Zea mays)/corn/soybean (Glycine max) (CCS), corn/soybean/corn (CSC), soybean/corn/soybean (SCS). Soybean yields were in decreasing order, CC>CCS>CSC>SCS, and the yield differences could not be explained by soil chemical and physical properties previously reported by a different study on the same sites. Our goal was to relate soil biological properties, including soil health indicators and soil microbial community composition, with the differences in soybean yields. To assess the soil health, soil protein, permanganate-oxidizable carbon (POXC) and β-glucosidase activity were measured. After 14 years of the above-mentioned rotation regimes, soil protein was significantly higher in continuous corn (CC) plots compared with other rotations (pChthoniobacter, and one taxa of fungi, Ascomycota, were associated with higher levels of β-glucosidase and correlated with the lower soybean yield observed in the SCS treatment. We also found a differential abundance of specific bacterial and fungal Operational Taxonomic Units (OTUs) to be informative on predicting yield differences, especially fungal pathogens from the genera Macrophomina and Corynespora at one location. Informative bacterial OTUs, however, are not pathogens, and belong to an uncultured family.