Periphyton in aquatic ecosystems is comprised of algae, heterotrophic microbes, and detritus. In shallow lakes, periphyton is affected by substrate type, nutrient supply, and other factors. Periphyton on macrophytes can interact strongly with the host, but whether different periphyton on non-plant substrates are affected by nearby submerged macrophytes is unclear. We conducted a six-month mesocosm study to investigate the responses of periphyton (on sand, cobblestone, or plastic) to different nitrogen concentrations (i.e., 10 mg L.1 ammonia, 10 mg L.1 nitrate, or no addition) in the presence of different macrophyte species (i.e., Vallisneria spinulosa, Potamogeton wrightii, and Potamogeton maackianus). Periphytic pigment content (chlorophyll and pheophytin), nutrient accrual (carbon, nitrogen, and phosphorus), carbon:chlorophyll ratio, and algal species composition were determined at the end of the experiment. Results showed that substrate type was critical for all periphyton parameters. Periphytic nutrient accrual and the carbon:chlorophyll ratio were both higher on sand than on the other two substrates. Nitrogen concentrations had a considerable effect on periphytic chlorophyll-a, nitrogen, and phosphorus accrual. In particular, the addition of ammonia (but not nitrate) led to significantly higher periphytic chlorophyll-a than the treatment without nitrogen. Furthermore, periphytic algal species richness and diversity were lower in the presence of P. wrightii than in the presence of the other macrophyte species. Thus, in addition to the importance of substrate type and nitrogen concentration, the species composition of nearby submerged macrophytes could potentially influence the periphyton community on a non-plant substrate.