Microplastics are frequently detected in freshwater environments, serving as a new factitious substrate for colonization of biofilm-forming microorganisms. Distinct microbial assemblages between microplastics and surrounding waters have been well documented; however, there is insufficient knowledge regarding biofilm colonization of plastic and non-plastic substrates, despite the fact that microbial communities generally aggregate on natural solid surfaces. In this study, the effects of substrate type on microbial communities were evaluated by incubation of biofilms on microplastic substrates (polyethylene and polypropylene) and natural substrates (cobblestone and wood) for 21 days under controlled conditions. Results from high-throughput sequencing of 16S rRNA revealed that the alpha diversity (richness, evenness, and diversity) was lower in the microplastic-associated communities than in those on the natural substrates, indicating substrate-type-coupled species sorting. Distinct community structure and biofilm composition were observed between these two substrate types. Significantly higher abundances of Pirellulaceae, Phycisphaerales, Cyclobacteriaceae, and Roseococcus were observed on the microplastic substrates compared with the natural substrates. Simultaneously, the functional profiles (KEGG) predicted by Tax4Fun showed that the pathways of amino acid metabolism and metabolism of cofactors and vitamins were increased in biofilms on the microplastic substrates. The findings illustrate that microplastic acts as a distinct microbial habitat (compared with natural substrates) that could not only change the community structure but also affect microbial functions, potentially impacting the ecological functions of microbial communities in aquatic ecosystems. Display omitted •Alpha diversity of biofilms was lower on microplastic than on natural substrates.•Community structure and composition varied between biofilms on different substrates.•Metabolic pathways were altered in biofilms colonizing microplastic.•Microplastic is a new microbial niche affecting microbial structure and function.•This alteration in biofilms may have an ecological impact on aquatic ecosystems.