Little information is known about the effects of different fertilization practices on soil microbiome in intensively managed crop rotations. The objective of this research was to investigate the response of microbial community composition (phospholipid fatty acid, PLFA) and extracellular enzyme activity to fertilization treatments through a three-year experiment. Treatments were: Control (without fertilizer, CK), chemical fertilizer (NPK), NPK + pig manure (NPKM), NPK + straw (NPKS), and NPK + both manure and straw (NPKMS). We found that fertilization had no effect on the microbial abundance except arbuscular mycorrhizal fungi (AMF) PLFA. Soil microbial community composition was significantly affected by crop species and to a lesser extent by fertilization, with a greater influence on the wheat harvest. In addition, soil enzyme activities were enhanced by fertilization, especially in wheat season. Over three years, compared with NPK treatment, addition of organic manure or straw (NPKS and NPKMS) significantly increased the activities of the enzymes except invertase and urease, and the effect was greater at wheat harvest than the maize harvest. Our results indicate that the response of soil microbial community structure and enzyme activities to fertilization takes precedence than microbial biomass in the short term. The temporal variation in soil microbial community structure and enzyme activities in the crop rotation indicate that crop species may be carefully considered for sustainable agricultural intensification management.