Excessive nitrogen fertilizer usage in agricultural often leads to negative ecological and production gains. Alterations in the physical and chemical properties and microbial community structure of agricultural soils are both the cause and consequence of this process. This study explored the perturbation of soil properties and microorganisms in agricultural soils by different nitrogen levels. Soil total nitrogen, total phosphorus, and total potassium decreased in the shallow soil layer with decreasing nitrogen. Changes in nitrogen affected soil organic matter, pH, bulk density, and water content. However, a moderate reduction in nitrogen did not cause significant yield loss; the increased nitrogen use efficiency was the main reason, attributed to the available phosphorus and potassium. Short-term changes in nitrogen had limited effects on soil microbial community structure. Bacteria were more susceptible to perturbation by nitrogen changes. Nitrogen reduction increased the relative abundance of MND1 (1.21%), RB41 (1.96%), and Sphingomonas (0.72%) and decreased Dongia (0.3%), Chaetomium (0.41%), and Penicillium (0.5%). Nitrogen reduction significantly increased the bacteria functional composition of aerobic ammonia oxidation (4.20%) and nitrification (4.10%) and reduced chemoheterotrophy (2.70%) and fermentation (4.08%). Available phosphorus specifically drove bacterial community structure variation in the shallow soil layers of moderate nitrogen reduction treatments. Steroidobacter, RB41, Gemmatimonas, Ellin6067, Haliangium, and Sphingomonas were the main component nodes in this community structure. These results provide insights into the study of nitrogen and microorganisms in agricultural soils.