The present study was performed to investigate the effects of NH3-N and nitrifying microorganisms on the increased BOD of downstream of the Yeongsan river in Gwangju. Water samples were collected periodically from the 13 sampling sites of rivers from April to October 2021 to monitor water qualities. In addition, the trends of nitrogenous biochemical oxygen demand (NBOD) and microbial clusters were analyzed by adding different NH3-N concentrations to the water samples. The monitoring results showed that NH3-N concentration in the Yeongsan river was 22 times increased after the inflow of discharged water from the Gwangju 1st public sewage treatment plant (G-1-PSTP). Increased NH3-N elevated NBOD levels through the nitrification process in the river, consequently, it would attribute to the increase of BOD in the Yeongsan river. Meanwhile, there was no proportional relation between NBOD and NH3-N concentrations. However, there was a significant difference in NBOD occurrence by sampling sites. Specifically, when 5 mg/L NH3-N was added, NBOD of the river sample showed 2-4 times higher values after the inflow of discharged water from G-1-PSTP. Therefore, it could be thought other factors such as microorganisms influence the elevated NBOD levels. Through next-generation sequencing analysis, nitrifying microorganisms such as Nitrosomonas, Nitroga, and Nitrospira (Genus) were detected in rivers samples, especially, the proportion of them was the highest in river samples after the inflow of discharged water from G-1-PSTP. These results indicated the effects of nitrifying microorganisms and NH3-N concentrations as important limiting factors on the increased NBOD levels in the rivers. Taken together, comprehensive strategies are needed not only to reduce the NH3-N concentration of discharged water but also to control discharged nitrifying microorganisms to effectively reduce the NBOD levels in the downstream of the Yeongsan river where discharged water from G-1-PSTP flows.