The study investigates the microbial mechanisms underlying the mineralization of soil organic matter (SOM) induced by straw in biochar-amended paddy soil. Straw return increases SOM content but also priming effects, which can reduce soil carbon sequestration. The addition of biochar, a byproduct of straw pyrolysis, is known to enhance microbial activity and SOM mineralization. The research uses 13C-labeled straw to trace carbon sources in mineralized CO2 and phospholipid fatty acids (PLFAs) of soil microbes. The study found that cumulative SOM mineralization increased by 61% and 27% in soils amended with Straw + Biochar (BC) and Straw + Ferrihydrite + BC, respectively, compared to straw-only treatments. Biochar addition increased the incorporation of 13C into PLFAs, reflecting accelerated microbial turnover. The microbial community composition changed with straw decomposition, with an increase in gram-positive bacteria (Gram+) and fungi, which are the main active microorganisms involved in SOM mineralization. Nutrient availability, particularly Olsen P, influenced microbial reproduction and growth. The results highlight that biochar acts as an electron shuttle, stimulates iron reduction, and releases organic carbon from soil minerals, thereby increasing SOM mineralization. Gram+ bacteria and fungi were crucial in straw decomposition and SOM mineralization under anaerobic conditions in the presence of biochar.The study investigates the microbial mechanisms underlying the mineralization of soil organic matter (SOM) induced by straw in biochar-amended paddy soil. Straw return increases SOM content but also priming effects, which can reduce soil carbon sequestration. The addition of biochar, a byproduct of straw pyrolysis, is known to enhance microbial activity and SOM mineralization. The research uses 13C-labeled straw to trace carbon sources in mineralized CO2 and phospholipid fatty acids (PLFAs) of soil microbes. The study found that cumulative SOM mineralization increased by 61% and 27% in soils amended with Straw + Biochar (BC) and Straw + Ferrihydrite + BC, respectively, compared to straw-only treatments. Biochar addition increased the incorporation of 13C into PLFAs, reflecting accelerated microbial turnover. The microbial community composition changed with straw decomposition, with an increase in gram-positive bacteria (Gram+) and fungi, which are the main active microorganisms involved in SOM mineralization. Nutrient availability, particularly Olsen P, influenced microbial reproduction and growth. The results highlight that biochar acts as an electron shuttle, stimulates iron reduction, and releases organic carbon from soil minerals, thereby increasing SOM mineralization. Gram+ bacteria and fungi were crucial in straw decomposition and SOM mineralization under anaerobic conditions in the presence of biochar.