In this study, the 16S rRNA-based Illumina MiSeq sequencing was used to investigate the bacterial community structure and composition of intertidal biofilm taken along the Yangtze Estuary. The results showed that 680,721 valid sequences of seven samples were assigned to 147,239 operational taxonomic units, which belonged to 49 phyla, 246 family and 314 genera. Compared to other studies on water and sediments in the study area, biofilms showed highest index of bacterial diversity and abundances. At different taxonomic levels, both dominant taxa and their abundances varied among the seven samples, with Proteobacteria as the dominant phylum in general. Principal component analysis and cluster analysis revealed that bacterial communities at WSK differed from those at other sampling sites. Salinity, dissolved oxygen, pH and nutrients were the vital environmental factors to influence the bacterial community structure of biofilms. These results may provide a new insight into the microbial ecology in estuarine environments.