Preventing the rebound of black and odorous water bodies is critical for improving the ecological environment of water bodies. This study examined the effect and underlying mechanism of in-situ improvement of the sediment microenvironment by nitrate in the tailwater of wastewater treatment plants combined with aerobic denitrifying bacteria under low-DO regulation (TailN + CFM + LDO). On the 60th day of remediation, the levels of dissolved oxygen and oxidation–reduction potential in the overlying water rose to 5.6 mg/L and 300 mV, respectively, the concentration of acid volatile sulfide within the sediment significantly decreased by 70.4%, and the organic matter content in the sediment was reduced by 62.7%, in which the heavy fraction organic matter was degraded from 105 g/kg to 56 g/kg, and the potential risk of water reverting to black and odorous conditions significantly decreased. Amplicon sequencing analysis revealed that the relative abundance of the electroactive bacteria Thiobacillus and Pseudomonas with denitrification capacity was found to be significantly higher in the TailN + CFM + LDO group than in the other remediation groups. Functional prediction of the 16S sequencing results indicated that both the quantity and activity of critical microbial enzymes involved in nitrification and denitrification processes could be enhanced in the TailN + CFM + LDO group. These results improved our understanding of the improvement of the sediment microenvironment and could thus facilitate its application.