Biofilms can colonize nearly any surface, and in aquatic ecosystems they are essential contributors to the biogeochemical cycles and food webs. Plastic waste that ends up in waterways is a new type of surface for biofilm colonization. To analyze the effects of plastic pollution on microbial freshwater biofilms, we incubated sheets of poplar wood (Liriodendron tulipifera) and high-density polyethylene (HDPE) with two veneer textures, smooth and rough, in three headwater streams at the Savannah River Site (Aiken, SC, USA). We found that plastic negatively impacts species richness and evenness, but patterns varied. For example, richness was greatest on wood sheets, and evenness was highest on rough HDPE. Also, beta diversity differed most between wood and plastic sheets and became more different over time. Our findings emphasize the need to further analyze plastic pollution effects on biofilms.