Ryegrass was pulse-labeled with enriched ¹³CO₂ for 18 h, followed by dynamic photosynthetic-carbon (¹³C) quantification in the plant (shoot, root), soil aggregates (three size classes), and microbial phospholipids fatty acids (PLFA-SIP) in soil amended with or without 700 °C-pyrolyzed biochar. We observed that biochar led to no difference of ¹³C allocation in shoot or root but reduced 88.7% of total ¹³C in soil, with decreased incorporation by 92.8% (macroaggregates), 94.5% (microaggregates), and 84.1% (silt-clays), respectively, compared to biochar-unamended soil. Meanwhile, biochar exerted negative effects on fungal relative abundance but led to positive impacts on that of bacteria, e.g., it reduced root-associated fungi (i.e., 16:1ω5c) and fungal-assimilated ¹³C (from averagely 71.2 ng C g⁻¹ soil to 26.3 ng C g⁻¹ soil after biochar application). The enhanced bacteria/fungi could be driven by biochar-mediated pH increase that relieved acid stress to bacteria. Co-occurrence network confirmed that biochar addition favored bacteria to compete with fungi, leading to decreased aggregation and stability (indicated by reduced normalized mean weight diameter) due to less fungal entangling with aggregates, thus exposing the rhizodeposits to bacterial (i.e., actinomycetes) decomposition. The correlation analysis further evidenced that fungal abundance was associated with ¹³C accumulation in soil aggregates, while bacterial relative abundance especially that of actinomycetes was negatively correlated with ¹³C accumulation. Random forest modeling (RF) supported the contributions of fungi to ¹³C-sequestration compared to bacteria. Taken together, we concluded that less stabilization of rhizodeposits in the biochar-amended soil was due to changes in microbial community, particularly the balance of fungi-bacteria and their interactions with soil physicochemical properties, i.e., aggregation and pH.