Submerged macrophytes play important roles in constructed wetlands and natural water bodies, as these organisms remove nutrients and provide large surfaces for biofilms, which are beneficial for nitrogen removal, particularly from submerged macrophyte-dominated water columns. However, information on the responses of biofilms to submerged macrophytes and nitrogen molecules is limited. In the present study, bacterial community structure and denitrifiers were investigated in biofilms on the leaves of four submerged macrophytes and artificial plants exposed to two nitrate concentrations. The biofilm cells were evenly distributed on artificial plants but appeared in microcolonies on the surfaces of submerged macrophytes. Proteobacteria was the most abundant phylum in all samples, accounting for 27.3–64.8% of the high-quality bacterial reads, followed by Chloroflexi (3.7–25.4%), Firmicutes (3.0–20.1%), Acidobacteria (2.7–15.7%), Actinobacteria (2.2–8.7%), Bacteroidetes (0.5–9.7%), and Verrucomicrobia (2.4–5.2%). Cluster analysis showed that bacterial community structure can be significantly different on macrophytes versus from those on artificial plants. Redundancy analysis showed that electrical conductivity and nitrate concentration were positively correlated with Shannon index and operational taxonomic unit (OTU) richness (log₁₀ transformed) but somewhat negatively correlated with microbial density. The relative abundances of five denitrifying genes were positively correlated with nitrate concentration and electrical conductivity but negatively correlated with dissolved oxygen.