Sustainable vegetative management plays a significant role in improving soil quality in degraded agricultural landscapes by enhancing soil microbial biomass. This study investigated the effects of grass buffers (GBs), biomass crops (BCs), grass waterways (GWWs), and agroforestry buffers (ABs) on soil microbial biomass and soil organic C (SOC) compared with continuous corn (Zea mays L.)–soybean [Glycine max (L.) Merr.] rotation (row crop [RC]) on claypan soils. The RC, AB, GB, GWW, and BC treatments were established in 1991, 1997, 1997, 1997, and 2012, respectively, and are located at Greenley Memorial Research Center in Missouri. Soil samples were collected in May 2018 from the 0‐ to 10‐cm depth at summit, backslope, and footslope landscape positions. Within AB treatment, soils were collected from the 50‐cm and 150‐cm tree distance. Total microbial biomass and biomass of gram‐positive bacteria, gram‐negative bacteria, actinomycetes, rhizobia, fungi, arbuscular mycorrhizae, saprophytes, and protozoa were determined by phospholipid fatty acid (PLFA) analysis. Results showed that soil microbial biomass and SOC across all microbial groups were significantly higher (P < .01) under perennial vegetation treatments compared with RC. The footslope position exhibited the highest total microbial biomass compared with the summit and backslope positions. The sampling distance of 50 cm from the tree base demonstrated 16% greater total microbial biomass and 15% higher SOC compared with 150 cm. These findings highlight the influence of landscape on soil biological properties and show that perennial vegetation systems have the potential to increase soil microbial biomass and enhance agricultural sustainability in degraded RC systems.