Lacustrine sediment played important roles in migration and transformation of its water soluble organic matter (WSOM), and the source and composition of WSOM would affect water trophic status and the fate of pollutants. However, we know little about the pathway of WSOM transformation and its driving bacterial communities in lacustrine sediment. In the present study, we investigated the spatial distribution patterns of sediment WSOM and its fluorescent fractions across Lake Chaohu using fluorescence spectroscopy, and explored WSOM compositional structure through our proposed calculated ratios. In addition, we also analyzed sediment bacterial community using Illumina sequencing technology, and probed the possible pathway of sediment WSOM transformation under the mediate of indigenous bacteria. Our results showed that the inflowing rivers affected the spatial distribution patterns of WSOM and its five fractions (including tyrosine-, tryptophan-, fulvic acid-, humic acid-like substances and soluble microbial productions), and sediment WSOM originated from fresh algae detritus or bacterial sources. In parallel, we also found that Proteobacteria (mainly γ-Proteobacteria and δ-Proteobacteria), Firmicutes (mainly Bacilli), Chloroflexi, Acidobacteria, Planctomycetes and Actinobacteria dominate sediment bacterial community. Furthermore, these dominant bacteria triggered sediment WSOM transformation, specifically, the humic acid-like substances could be converted into fulvic acid-like substances, and further degraded into aromatic protein-like and SMP substances. In addition, our proposed ratios (P-L:H-L, Ar-P:SMP and H-L ratio), as supplementary tool, were effective to reveal WSOM composition structure. These results figured out possible pathway of WSOM transformation, and revealed its microbial mechanism in lacustrine sediment.