Intercropping kura clover (Trifolium ambiguum) (KC) with prairie cordgrass (Spartina pectinata) (PCG) has great potential for biofuel feedstock on marginal lands. This study evaluated the impacts of 10‐year PCG‐KC intercropping and PCG monoculture fertilized with different nitrogen (N) rates of granular urea (five treatments: PCG‐KC, PCG‐0N, PCG‐75N, PCG‐150N, and PCG‐225N) on soil biogeochemical properties: (i) in the surface soil (0‐ to 10‐cm depth) at three different sampling times during the crop growing season: spring (April, pre‐emergence), summer (June, active growth), and fall (November, post‐harvest); and (ii) at different soil depths (0–5, 5–15, 15–30, 30–45, and 45–60 cm) (only total carbon (C) and N) in fall 2021. All soil biogeochemical parameters were higher during summer as compared to spring and/or fall, except urease activity, ammonium‐N, microbial biomass C and N, and fluorescein diacetate (FDA). On average over the sampling times, PCG‐KC had significantly higher β‐glucosidase activity and hot‐water extractable organic N than PCG‐0N; but no significant difference between PCG‐KC and N‐fertilized PCG. Cold‐water extractable organic N was significantly lower than the highest N rate, but not significantly different from PCG‐0N and lower N rate treatments. Urease activity under PCG‐KC treatment was double that of PCG‐0N and PCG‐75N; FDA was higher in PCG‐KC than all monocultures. No treatment effect was found on soil total C and N, except that they decreased with depth. Overall, intercropping PCG‐KC showed some benefits in terms of promoting soil biogeochemical properties during crop growth periods, having lower residual reactive N in the soil, and maintaining biomass yield and quality on marginal lands.