Soil organic carbon (SOC) is pivotal for both agricultural activities and climate change mitigation, and biochar stands as a promising tool for bolstering SOC and curtailing soil carbon dioxide (CO2) emissions. However, the involvement of biochar in SOC dynamics and the underlying interactions among biochar, soil microbes, iron minerals, and fresh organic matter (FOM, such as plant debris) remain largely unknown, especially in agricultural soils after long-term biochar amendment. We therefore introduced FOM to soils with and without a decade-long history of biochar amendment, performed soil microcosm incubations, and evaluated carbon and iron dynamics as well as microbial properties. Biochar amendment resulted in 2-fold SOC accrual over a decade and attenuated FOM-induced CO2 emissions by approximately 11% during a 56-day incubation through diverse pathways. Notably, biochar facilitated microbially driven iron reduction and subsequent Fenton-like reactions, potentially having enhanced microbial extracellular electron transfer and the carbon use efficiency in the long run. Throughout iron cycling processes, physical protection by minerals could contribute to both microbial carbon accumulation and plant debris preservation, alongside direct adsorption and occlusion of SOC by biochar particles. Furthermore, soil slurry experiments, with sterilization and ferrous iron stimulation controls, confirmed the role of microbes in hydroxyl radical generation and biotic carbon sequestration in biochar-amended soils. Overall, our study sheds light on the intricate biotic and abiotic mechanisms governing carbon dynamics in long-term biochar-amended upland soils.