Soil aggregates play a crucial role in microbially mediated soil organic carbon (SOC) cycling. However, the microbial constraints governing SOC storage under straw incorporation at different soil aggregation levels remain unclear. To address this, a 4-year field experiment was conducted in northeast China using a completely randomized block design with three replicates: conventional tillage (15 cm depth) without straw (CT) and with straw (10,000 kg ha−1 y−1, SCT), as well as deep tillage (35 cm depth) without straw (DT) and with straw (10,000 kg ha−1 y−1, SDT). Soil aggregates were fractionated into > 2 mm, 2–0.25 mm, and < 0.25 mm size classes. Compared to CT, SCT, and DT, the SOC storage under SDT increased significantly by 4.45 %, 1.71 %, 4.64 %, respectively, across the 0–35 cm soil depth. Straw incorporation (SCT vs. CT) elevated the proportion of > 2 mm and 2–0.25 mm aggregates, along with soil organic carbon, nitrogen, phosphorus, potassium nutrient contents, and microbial biomass within the 0–15 cm layer. Similarly, SDT (vs. DT) enhanced these parameters in both the 0–15 cm and 15–35 cm layers. Straw addition (SCT vs. CT, SDT vs. DT) also increased the microbial diversity (Actinobacteriota and Chloroflexi in the 0–15 cm layer), and complexity of microbial co-occurrence networks in 2–0.25 mm and < 0.25 mm aggregates while alleviating microbial carbon (by 0.93–2.29 %) and phosphorus (by 0.80–2.94 %) limitations. Partial least squares path modeling indicated that the 2–0.25 mm aggregate fraction was the primary driver of SOC storage enhancement under straw incorporation, with bacterial and fungal co-occurrence network relationships collectively mitigating microbial resource (carbon and phosphorus) limitations in this key size class. Therefore, these findings underscore the importance of microbial regulation in SOC dynamics at the aggregate level, providing insights for field management strategies aimed at improving soil quality.