Biochar is a beneficial substance for sequestering organic carbon in soil. Application of fresh biochar (FB) to the soil leads to an increase in soil organic carbon (SOC). However, the effects of aged biochar on SOC stabilization in acidic soils are unclear. A 90‐day incubation study was assessed to evaluate the impact of adding rice straw biochar on acid soil. The study aimed to assess the alterations in carbon fractions, enzyme activity and bacterial communities in soil. We used different ageing processes (water‐washed biochar [WB] and H2O2 oxidation biochar [OXB]) at a rate of 2%, with control at 0% of biochar. The results indicated that aged biochar reduced CO2 efflux and increased microbial biomass carbon (MBC), reducing the microbial metabolic quotient (qCO2). The SOC and its fractions increased with ageing relative to the FB treatment. Compared with the FB treatment, the carbon pool management index (CPMI) also increased with the OXB application. Additionally, the application of OXB stimulated the soil enzymes involved in carbon cycling, specifically β‐1,4‐glucosidase (βG), β‐1,4‐xylosidase (βX) and cellobiohydrolase (CBH) relative to the FB and WB treatments. Moreover, biochar applications improved the structure and functions of the bacterial population in soil with high acidity. Proteobacteria, Acidobacteria, Actinobacteria and Chloroflexi were the most prevalent species in all treatments, as shown by their relative abundances. The redundancy analysis revealed that the DOC (dissolved organic carbon) and MBC significantly impacted on the microbial community. Furthermore, adding OXB altered the Bacterial community involved in the C cycle by affecting SOC. These findings help us learn more about how applying fresh or aged rice straw biochar changes different carbon fractions and microbial communities in soil. It may serve as an instance for studying the provision of soil organic carbon after applying biochar in acidic soil.