Abstract Converting crop residues into biochar and subsequently incorporating it into soils is a promising strategy to improve carbon sequestration in agroecosystems. However, differences in the potential mechanisms for soil organic carbon (SOC) accumulation between biochar and its feedstock materials remain unclear. This study performed a nine-year field experiment involving four treatments in a Mollisol: mineral fertilizers alone and combined with maize straw or its biochar, and control without fertilizers and amendments. The main objectives of this study were to assess how straw and biochar incorporation influence SOC content, SOC chemical composition, microbial necromass carbon (MNC) content, and microbial community characteristics. Compared with mineral fertilizers alone, straw and biochar significantly ( P < 0.05) increased SOC content, while biochar (40.5% increase) had more pronounced effects than straw (23.7% increase). Similarly, both straw and biochar increased MNC content and boosted microbial biomass, but straw had a more pronounced impact than did biochar. Additionally, biochar markedly increased SOC aromaticity by 26.6% ( P < 0.05), while straw reduced SOC aromaticity by 2.85% ( P > 0.05). Fungi, rather than bacteria, played a more crucial role in increasing SOC content through their necromass inputs under both straw and biochar incorporation. Our results demonstrated that straw enhanced SOC accumulation primarily through entombing effect of microbial necromass, while biochar enhanced SOC accumulation primarily through biochemical protection from recalcitrant aromatic carbon. The present findings provide valuable insight into the potential mechanisms of field practices that lead to maximize soil carbon sequestration. Graphical Abstract