This study investigates the microbial mechanisms of organic matter mineralization induced by straw in biochar-amended paddy soils. Straw return increases soil organic matter (SOM) content but also primes SOM mineralization. Biochar application enhances microbial activity and SOM mineralization by acting as an electron shuttle, stimulating iron reduction, and releasing organic carbon from soil minerals. The study found that biochar-amended soils showed increased SOM mineralization compared to straw-only soils, with a 61% increase in the Straw + BC treatment and a 27% increase in the Straw + Fh + BC treatment. The microbial community composition changed with biochar addition, increasing the abundance of 13C-labeled gram-positive bacteria (13C-Gram+) and fungi, which are the main active microorganisms involved in SOM mineralization. The 13C incorporation into microbial biomass and phospholipid fatty acids (PLFAs) was higher in biochar-amended soils, indicating accelerated microbial turnover. The study also found that microbial reproduction and growth were influenced by nutrient availability, with 13C-Gram+ and 13C-fungi increasing with Olsen P but decreasing with dissolved organic carbon and NO3- contents. Overall, biochar application enhances microbial activity and SOM mineralization by promoting the growth of gram-positive bacteria and fungi, which are key drivers of SOM mineralization in paddy soils. The study highlights the importance of active microbial taxa, particularly gram-positive bacteria and fungi, in anaerobic iron-enriched soils mixed with biochar and straw, and their contribution to SOM mineralization.This study investigates the microbial mechanisms of organic matter mineralization induced by straw in biochar-amended paddy soils. Straw return increases soil organic matter (SOM) content but also primes SOM mineralization. Biochar application enhances microbial activity and SOM mineralization by acting as an electron shuttle, stimulating iron reduction, and releasing organic carbon from soil minerals. The study found that biochar-amended soils showed increased SOM mineralization compared to straw-only soils, with a 61% increase in the Straw + BC treatment and a 27% increase in the Straw + Fh + BC treatment. The microbial community composition changed with biochar addition, increasing the abundance of 13C-labeled gram-positive bacteria (13C-Gram+) and fungi, which are the main active microorganisms involved in SOM mineralization. The 13C incorporation into microbial biomass and phospholipid fatty acids (PLFAs) was higher in biochar-amended soils, indicating accelerated microbial turnover. The study also found that microbial reproduction and growth were influenced by nutrient availability, with 13C-Gram+ and 13C-fungi increasing with Olsen P but decreasing with dissolved organic carbon and NO3- contents. Overall, biochar application enhances microbial activity and SOM mineralization by promoting the growth of gram-positive bacteria and fungi, which are key drivers of SOM mineralization in paddy soils. The study highlights the importance of active microbial taxa, particularly gram-positive bacteria and fungi, in anaerobic iron-enriched soils mixed with biochar and straw, and their contribution to SOM mineralization.