The specific freshwater environment of reservoirs formed by streams has not been well studied. In this paper, the bacterioplankton community in such a reservoir, the Huangqian Reservoir in eastern China, was described using culture-independent molecular methods. We found that the most dominant bacterioplankton were affiliated with Cyanobacteria, followed by Betaproteobacteria, Bacteroidetes, Gammaproteobacteria, and Actinobacteria. Both bacterial abundance and diversity increased along the direction of water flow, and the 16S rRNA gene copy number in the water outlet was nearly an order of magnitude higher than that in the water inlet. Pearson correlation analyses indicated that nitrate had a significantly negative correlation with the bacterial abundance (p < 0.05) and that ammonium was positively correlated with bacterial abundance (p < 0.05). Interestingly, owing to a remarkably negative correlation (p < 0.01), the ratio of nitrate and ammonium might serve as a good pre dictor of the relative abundance of bacterioplankton. According to redundancy analysis, nitrate and dissolved oxygen were the major factors influencing the bacterial communities. In addition, we attempted to determine the reasons why such a reservoir could maintain good ecological balance for a period of decades, and we found that the environmental factors and bacterial communities both played critical roles. This research will benefit our understanding of bacterial communities and their surrounding environments in freshwater ecosystems.