Radial oxygen loss (ROL) has been suggested to be a major process to protect plants exposed to the anaerobic by-products of soil anaerobiosis. The aim of the present study was to test the effects of root ROL from two submerged plants (Hydrilla verticillata and Vallisneria spiralis) on the rhizosphere oxygen profile and rhizosphere microarea. Phospholipid fatty acids (PLFAs) of sediment samples were used to characterize and quantify the microbial community. The results showed clearly that there were significant differences between the two plants in radial oxygen loss, which affected rhizosphere physicochemical parameters and the microbial community. Rhizosphere total biomass, bacteria, gram-positive bacteria, actinomycetes, and microbial diversity of V. spiralis were significantly higher than those of H. verticillata. The present study highlights root ROL as a key parameter affecting the microbial community of the rhizosphere microarea.