The objective of this paper was to study the biofilm microbial communities from Qinhuai River, Stone town moat, Lake Wulong and Lake Xuanwu of Nanjing using phospholipid fatty acid (PLFA) method. (1) It was found that the decreasing of temperature and nutrients concentration led to the slowing down of the metabolism rate and even the death of microorganisms. The microbial biomass ranged from 9.16×10⁶ to 2.74×10⁸ cells/g in static water, while in running water, it ranged from 1.69×10⁷ to 7.77×10⁷cells/g. The decline of microbial biomass in static water could be attributed to the higher transfer resistance. (2) G-, G+ and fungi mainly comprised the microbial community in the order:G- > fungi > G+. As temperature decreasing (T ≥ 8.4℃), the content of functional bacteria in static water fluctuated from 27.41% to 66.20%, while in running water it fluctuated from 43.09% to 68.25%. The difference might be caused by G- and fungi. (3) In static water, temperature, concentration of nitrate and COD largely influenced the growth of functional bacteria, while in running water the concentration of nitrogen was critical.