The toxic eﬀects of nanoparticles on individual organisms have been widely investigated, while few studies have investigated the eﬀects of nanoparticles on ubiquitous multi-community microbial aggregates. Here, periphyton as a model of microbial aggregates, was employed to investigate the responses of microbial aggregates exposed continuously to Fe2O3 nanoparticles (5.0 mg L−1) for 30 days. The exposure to Fe2O3 nanoparticles results in the chlorophyll (a, b, and c) contents of periphyton increasing and the total antioxidant capacity decreasing. The composition of the periphyton markedly changes in the presence of Fe2O3 nanoparticles and the species diversity signiﬁcantly increases. The changes in the periphyton composition and diversity were due to allelochemicals, such as 3-methylpentane, released by members of the periphyton which inhibit their competitors. The functions of the periphyton represented by metabolic capability and contaminant (organic matter, nitrogen, phosphorus and copper) removal were able to acclimate to the Fe2O3 nanoparticles exposure via self-regulation of morphology, species composition and diversity. These ﬁndings highlight the importance of both physiological and ecological factors in evaluating the long-term responses of microbial aggregates exposed to nanoparticles.