Microbial communities in biofilms have been suggested to mediate a wide spectrum of ecological functions. However, the underlying mechanism of most of the mediations is still unresolved mainly because of the uncertainty in characterizing members in the communities using existing 16S rRNA gene-based molecular methods. In this study, a newly developed method – barcoded 16S rRNA gene pyrosequencing – was employed to provide a detailed characterization of the bacterial communities in intertidal and subtidal marine biofilms developed in two seasons. This method has high sensitivity to detect low-abundance taxa and is able to generate a large amount of highly reliable data in a very short period of time. Our results revealed highly diverse biofilm bacterial communities varied with season and tidal level. Over 6,000 OTUs with species estimates of up to 15,000 were recovered in a biofilm sample, which is by far the highest record in sub-tropical marine biofilms and is much greater than biofilms developed in certain natural habitats. Nineteen phyla were found in the biofilm samples, for which Cyanobacteria and Proteobacteria were the most abundant phylum dominating the intertidal and subtidal biofilms, respectively. Apart from these two phyla, Actinobacteria, Bacteroidetes, and Planctomycetes were found to be the major groups recovered in both intertidal and subtidal biofilms, yet their relative abundance varied among samples. Full-length 16S rRNA gene clone library was constructed for the 4 biofilm samples and showed similar results with pyrosequencing. The relationship of bacterial community and settlement of larvae of a marine invertebrate is discussed.