Human activities have a tremendous impact not only on the macroscopic world, but also on the micro-organisms. Here, Amplified Ribosomal DNA Restriction Analysis (ARDRA) was used for assessing the effect of the industrial sewage on the microbial community in sludge of Dongting Lake, the second-largest freshwater lake in China. The sludge samples from the outfall of the representative nitrogenous fertilizer plant near the lake were collected in March, 2010, and the sludge samples from the surrounding waters were treated as the control. The multi-element analysis results showed that the content of nitrogen, phosphorus in Sample SY were 1.9 and 1.47 times of the control group respectively. Based on restriction patterns derived from ARDRA, 26 representative clones (15 clones in the SY group and 11 clones in the DZ group) were sequenced. The sequence data and phylogenetic analysis of 16S rRNA gene presented that microorganism diversity of two sludge samples were abundant. Bacterial diversity presented among the outfall samples was dominated by Aeromonas sp. (5.8%), Acidimicrobidae sp. (5.8%) and Gemmatimonas sp. (5.0%). In contrast, bacterial diversity presented among the control group was dominated by Xanthomonas sp. (8.0%), Lautropia sp. (5.8%) and Duganella sp. (5.1%). The results indicated that due to the excessive of nitrogen and phosphorus discharged by the nitrogen fertilizer plant, the eutrophication in Dongting Lake has great influence on the microbial community structure.