Twelve sampling sites from two basins of Lake Chaohu were studied seasonally from June 2020 to April 2021 in Hefei City (China) to better understand the effect of organic carbon (C) quantity and composition on nitrate (NO3--N) reduction pathways. Serious algal bloom in the west basin of Lake Chaohu (WLC) resulted in higher organic C accumulation and NO3--N deficiency in interstitial water compared to the east basin of Lake Chaohu (ELC), jointly leading to a high C/NO3--N ratio. This triggered dissimilatory nitrate reduction to ammonium (DNRA) over denitrification in terms of higher DNRA rate, nitrogen retaining index (NRI), and nrfA gene abundance mediating DNRA. Furthermore, high oxygen-alkyl C and abundance of functional genes mediating labile organic C decomposition and DNRA suggested that the alkyl carbon-oxygen bond was responsible for DNRA induction. Different bacterial community composition and diversity involved in C and nitrogen (N) metabolism in two basins indicated that bacteria in sediments of WLC were more active in NO3--N reduction. Spearman correlation analysis showed that the less represented genera, such as Thiobacillus and Clostridium, were positively correlated with both organic C and NO3--N reduction rates, respectively. Hence, organic C composition could affect NO3--N reduction function by shaping the specific bacterial community.