The soil microbial community is sensitive to the ecological environment, and it can indicate changes in soil quality and determine the ecological functions of the soil. Phospholipid-derived fatty acids (PLFA) analysis was used to study the response of soil microorganisms to different configuration modes of sloping crop-mulberry systems on purple soil. The results showed that planting mulberry hedges significantly increased PLFA levels and improved the soil microbial community structure, when compared with no hedge control. The total PLFA level after T1 treatment (two belts of contour mulberry) was 20.54 nmol/g, which was 66.99% higher than the control. The number of soil bacteria after T1 treatment also increased significantly. The richness of fungi after T5 treatment (two belts of vertical mulberry) was relatively higher than that after the other treatments. The diversity and evenness indexes after T1 treatment and the richness index after T2 treatment (three belts of contour mulberry) were the highest among the treatments. Principal component (PC) analysis showed that 58.2% of PC1 was composed of straight-chain saturated and polyunsaturated fatty acids of a17:0, 16:1ω5c, and 17:0, while 77.5% of PC2 was composed of monounsaturated and cyclo fatty acids of i12:03OH, 20:1ω9c, and cy19:0ω8c. T1 and T2 treatments resulted in similar soil microorganisms that differed from the control. Redundancy analysis showed that the microbial community structure was mainly affected by total soil phosphorus, pH, total nitrogen, organic matter, and soil nitrate content. G+, branched saturated fatty acids, and bacteria were mainly affected by total phosphorus and total nitrogen, whereas G-was mainly influenced by soil pH. The amount of runoff was significantly and positively correlated with i16:0, 10Me17:0, a17:0, and 18:3ω6c (6,9,12) at P < 0.05 and positively correlated with 20:1ω9c at P < 0.001. The amount of sediments had a significant and positive correlation with 18:0, i16:0, and i173ω6c (6,9,12) at P < 0.05 and with 20:1ω9c at P < 0.001, whereas it had a significant and negative correlation with 18:3ω6c (6,9,12) at P < 0.05.