Switchgrass (Panicum virgatum L.) is usually grown on marginal land for biofuel system, in which nitrogen (N) is an essential management practice, and landscape position is a key topographical factor in impacting the production. However, limited information is available regarding how the N application and landscape positions affect soil microbial communities and enzyme activities under switchgrass. Thus, the specific objective of this study was to evaluate the responses of N rate (high, 112 kg N/ha; medium, 56 kg N/ha; and low, 0 kg N/ha) and landscape positions (shoulder and footslope) on soil biological health under switchgrass field. Data showed that N addition significantly influenced carbon and N fractions. The hot water‐soluble organic carbon (HWC) and nitrogen (HWN) fractions were significantly higher at footslope position than the shoulder position. The amount of total phospholipid fatty acid (PLFA), total bacterial, actinomycetes, gram‐negative and gram‐positive bacteria, total fungi, arbuscular mycorrhizal (AM) fungi, and saprophytes PLFAs were highest with medium and high N rates and footslope position. The N addition increased total PLFAs in N fertilizer treatments, viz. medium (5,946 ng PLFA‐C/g soil) and high N rates (5,871 ng PLFA‐C/g soil). Microbial biomass carbon and nitrogen and enzyme activities (urease, β‐glucosidase, acid phosphatase and arylsulfatase) were significantly enhanced by N fertilization (medium and high N rates) compared to control (low N rates) under footslope position. The urease activity under medium (36.3 µmol N‐NH4+ g−1 soil hr−1) and high N rates (31.4 µmol N‐NH4+ g−1 soil hr−1) was 42.9% and 23.6% higher than low N rates, respectively. This study suggests that the application of medium N rate in footslope position to switchgrass can enhance the soil biological properties and hence can protect the environment from the excessive use of N fertilizer.