Examining the soil microbiome structure has great significance in terms of exploring the mechanism behind plant growth changes due to maize (Zea mays L.) and soybean (Glycine max Merr.) crop rotation. This study explored the effects of soil microbial community structure after soybean and maize crop rotation by designing nine treatments combining three crop rotations (continuous cropping maize or soybean; and maize after soybean) with three fertility treatments (organic compound fertilizer, chemical fertilizer, or without fertilizer). Soil was sampled to 30 cm depth the second year at approximately the middle of the growing season, and was analyzed for physical, chemical, and phospholipid fatty acid (PLFA) profiles. Bacteria was found to be the predominant component of soil microorganisms, which mainly contained the PLFAs 16:0. Crop rotation with organic compound fertilizer application reduced the percentage of fungi in the soil by 24% compared to continuous maize and soybean with the same fertilizer application. The combination of crop rotation with organic fertilizer can reduce the percentage of fungi/bacteria to the greatest degree. In addition, the content of soil aggregate and organic matter had great influence on Gram-positive bacteria and actinomyces. In conclusion, soybean and maize crop rotation improve the soil nutrient content primarily by influencing the composition of bacterial community, especially the Gram-positive bacteria.