This study investigated biofilm establishment, biofilm structure, and microbial community composition of biofilms in three laboratory-scale moving bed biofilm reactors. These reactors were filled with three types of plastic carriers with varied depths of living space for microbial growth. The reactors were operated under the same influent and operational conditions. Along with the operation, the results showed that carriers with grids of 50 μm in height delayed the biofilm development and formed the thinnest biofilm and a carpet-like structure with the lowest α-diversity. In comparison, another two carriers with grids of 200 and 400 μm in height formed thick biofilms and large colonies with more voids and channels. Quantified properties of biofilm thickness, biomass, heterogeneity, portion of the biofilm exposed to the nutrient, and maximum diffusion distance were examined, and the results demonstrated that they almost (except for heterogeneity) strongly correlated to the α-diversity of microbial community. These illustrate that depth of living space, as an important parameter for carrier, could drive the formation of biofilm structure and community composition. It improves understanding of influencing factors on biofilm establishment, structure and its microbial community, and would be helpful for the design of biofilm processes.