Examining the soil microbiome structure has a great significance in 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 contain the PLFAs i15:0, 16:1 ω 7c, 16:0, 10Me16:0, and 18:1 ω 7c. The concentration of soil gram-negative bacteria from the soybean and maize rotation was less than in soybean continuous cropping when organic fertilizer was applied to both. Crop rotation reduced the percentage of fungi in the soil, among which the effect of organic compound fertilizer application was significantly reduced 24%. The combined crop rotation with organic fertilizer can reduce maximum the percentage of fungi/bacteria. In addition, the content of soil aggregate and organic matter had great influence on gram-positive bacteria and actinomyces, and soil pH had a greater impact on other fungi.