Purpose <p>Despite the recognized importance of dissolved organic matter (DOM) in soil carbon cycling, the molecular complexity and pronounced vertical heterogeneity of DOM have hindered a mechanistic understanding of how depth-structured DOM is associated with bacterial community organization in paddy soils.</p> Materials and methods <p>Here, we combined high-resolution mass spectrometry and 16S rRNA gene sequencing to investigate linkages between DOM molecular traits and bacterial composition across soil layers (0–20, 20–40, and 40–60 cm) in four representative paddy sites in South China.</p> Results and discussion <p>DOM molecular numbers decreased markedly with depth (on average from 2,848 formulas at 0–20 cm to 1,854 formulas at 40–60 cm), accompanied by a consistent decline in DOM chemodiversity (from 7.13 to 7.08). The middle soil layer functioned as a chemical transition zone, where intensified sulfur-associated transformations enhanced DOM condensation and aromaticity. These molecular changes coincided with decreased bacterial Shannon and Chao1 indies, reduced co-occurrence network connectivity (average degree decreased from 30.83 to 24.44), and a shift from oxidative metabolism toward fermentation-dominated functions. Random forest analysis identified lipid- and protein/amino sugar-like compounds as key predictors of bacterial diversity. However, their enrichment in deeper soil layers reflected the accumulation of refractory compounds with limited microbial accessibility rather than an increase in bioavailable substrates, thereby constraining microbial niche differentiation.</p> Conclusions <p>These findings provide insight into how depth-structured DOM covaries with microbial ecology in paddy soils and offer a foundation for improving predictions of soil carbon cycling under long-term agricultural management.</p> Graphical abstract <p></p>