Membrane fouling is the bottleneck of stable operations for reverse osmosis (RO), which is the key technology for reclaimed water reuse in thermal power plants. The foulant composition, formation mechanism, and key contributors were analyzed in this study. The primary scaling substances are Ca, Mg, Al, Fe, and Si, and Ca accounts for 1.49 wt%. Humic substances, proteins, and polysaccharides are the primary organic constitutes, and among them bio-derivatives were main composition revealed by FTIR, EEM, and SEC. Thus, bio-fouling was believed to be the key contributor together with the results of the SEM and the microbial community analysis. The three leading bacteria were alpha-, beta-, gamma-proteobacteria at class levels, 38.11%, 14.19%, and 34.31%, respectively, while starkeya, acidovorax, luteimonas, and pseudoxanthomonas were the leading bacteria at the genus level. Among these bacteria, the one with metabolic processes related to nitrogen fixation and proteolysis showed higher abundance for the high concentrations of nitrogen and protein. Foulants on the endcap and membrane entrance also indicated severe bio-fouling, where Acidovorax presented significant abundance. A Vertical distribution of the microbial community was found in the cross-section of the foulant, in particular, a significant decrease of starkeya (from 48.4% to 4.65%) and acidovorax (from 17.64% to 5.61%) and an increase of pseudoxanthomonas (from 1.04% to 12.89%) from the top layer to the bottom layer were observed. The gamma-proteobacteria was recognized as the pioneering bacteria for its significantly higher abundance in the deeper layer. This study helps to elucidate the RO membrane fouling composition and its key contributors, and improves our understanding of the membrane fouling mechanism and a controlling strategy.