To gain insights into the spatial pattern of microbial communities in Poyang Lake, we collected a total of 42 sediment samples from each of the four major parts of the lake in October 2010. We analyzed the spatial patterns of microbial biomass and microbial community composition using the phospholipid fatty acids (PLFA) method. The mean total microbial biomass was 2.28 mg PLFA/kg dry weight (wt) ranging from 0.25 to 6.56 mg PLFA/kg dry wt, indicating a high-spatial variation. The coefficients of variations (CVs) of the bacterial and fungal biomasses were 73 and 80%, respectively. In addition, the CVs of biomasses for the seven bacterial groups ranged from 62 to 92%, which included anaerobic bacteria, Gram-positive bacteria (Gþ), Gram-negative bacteria (GÀ), aerobic bacteria (AE), actinomycetes (A), sulfate-reducing bacteria (S), and methane-oxidizing bacteria. In our study, the ammonium concentrations both in the water and in the sediment were closely correlated to the total microbial biomass and the biomasses of bacterial groups, while the ratios of fungal to bacterial biomass (F/B) and GÀ/Gþ were correlated with ammonium concentration only in the water. In addition, F/B in the lake sediment was significantly correlated with the nitrogen content and the ratio of organic carbon to total nitrogen contents in the sediment (C/N). Moreover, the ammonium concentrations in the water and in the sediment, water dissolved organic carbon (DOC), sediment nitrogen content, and sediment organic carbon content were strongly linked to the spatial variation of bacterial community composition. Our results show that the C and N availability in the water and in the sediment are key predictors for microbial biomass and microbial community composition in Poyang Lake.