Soil microbes are an important component of terrestrial ecosystems connecting energy flows and matter cycles between the atmosphere and the soil. They play crucial roles in maintaining ecosystem function by driving a variety of complex ecological processes in soil. Here we report a field experiment on Stipa baicalensis steppe in the Inner Mongolia. The phospholipid fatty acids (PLFA) method was used to explore the effects of long-term different nutrient treatments (i.e. N, P, K in isolation and NP, NK, PK, and NPK in combination) on soil physical and chemical properties and microbial community structure. The results showed that the long-term nutrient addition did not significantly alter soil organic carbon and total nitrogen content, while P addition (P, NP, PK, NPK) and K addition (K, NK, PK, and NPK) significantly increased soil available phosphorus and available potassium content, respectively (P < 0.05). Single-N addition significantly increased soil ammonium nitrogen and nitrate nitrogen contents, but soil pH value decreased significantly. To a certain extent, Single-P and Single-K addition enhanced the total PLFA contents, the PLFA contents of bacteria, fungi, and actinomycetes. The Single-N and Multiple nutrient (NP, NK, PK, and NPK) addition significantly decreased the total PLFA contents, the PLFA contents of bacteria, fungi and actinomycetes. There were no significant differences under different nutrient addition treatments for the PLFA ratio of Gram-positive bacteria and Gram-negative bacteria (G⁺/G^-), while multiple nutrient (NP, NK and NPK) significantly decreased the PLFA ratio of fungi and bacteria (F/B). Correlation analysis indicated that soil microbial groups were positively correlated with soil available phosphorus and ammonium nitrogen content, and soil pH were negatively correlated with soil microbial groups. Redundancy analysis (RDA) and Random Forest (RF) analysis showed that soil pH and phosphorus content were found to be significant driving factor affecting soil microbial community structure. In summary, the long-term nutrient addition significantly changed soil available nutrients and soil pH, and then affected the microbial community structure.