AbstractImproving soil structure, fertility, and production is of major concern for establishing sustainable agroecosystems. Further research is needed to evaluate whether different methods of straw returning determine the variations of soil aggregation and the microbial community in aggregates in the long term. In this study, we comparatively investigated the effects of long-term fertilization regimes performed over six years, namely, non-fertilization (CK), chemical fertilization (CF), continuous straw return (CS), and continuous straw-derived biochar amendment (CB), on soil aggregation and bacterial communities in rice-wheat rotation systems. The results showed that straw/biochar application increased soil nutrient content and soil aggregate size distribution and stability at both 0–20 cm and 20–40 cm soil depths, compared with those of CF and CK; CB performed better than CS. CB increased bacterial community diversity and richness in 0–20 cm soil, and evenness in 0–40 cm soil (p < 0.05); CS had no significant effect on these aspects. Variations in the relative abundance of Actinobacteria, Chloroflexi, Bacteroidetes, Nitrospirae, Gemmatimonadetes, and Latescibacteria in specific aggregates confirmed the different effects of straw/biochar on bacterial community structure. The partial least squares discrimination analysis and permutation multivariate analysis of variance revealed that fertilization, aggregate size fractions, and soil depth affected the bacterial community, although their effects differed. This study suggests that CB may reduce chemical fertilizer usage and improve the sustainability of rice-wheat cropping systems over the long term, with a better overall outcome than CS.