This study investigates the presence and microbial colonization of microplastic in an urban river in Chicago, USA. Microplastic concentrations were found to be higher downstream of a wastewater treatment plant (WWTP) effluent compared to upstream, indicating that WWTP effluent is a significant source of microplastic in the river. High-throughput sequencing revealed that bacterial assemblages colonizing microplastic were less diverse and significantly different in taxonomic composition compared to those in the water column and suspended organic matter. Several taxa, including plastic-degrading organisms and pathogens, were more abundant on microplastic. The results suggest that microplastic in rivers is a distinct microbial habitat and may serve as a novel vector for the downstream transport of unique bacterial assemblages. Urban rivers, which receive high concentrations of microplastic from WWTP effluent, could play a significant role in the global microplastic life cycle and may have important ecological implications.This study investigates the presence and microbial colonization of microplastic in an urban river in Chicago, USA. Microplastic concentrations were found to be higher downstream of a wastewater treatment plant (WWTP) effluent compared to upstream, indicating that WWTP effluent is a significant source of microplastic in the river. High-throughput sequencing revealed that bacterial assemblages colonizing microplastic were less diverse and significantly different in taxonomic composition compared to those in the water column and suspended organic matter. Several taxa, including plastic-degrading organisms and pathogens, were more abundant on microplastic. The results suggest that microplastic in rivers is a distinct microbial habitat and may serve as a novel vector for the downstream transport of unique bacterial assemblages. Urban rivers, which receive high concentrations of microplastic from WWTP effluent, could play a significant role in the global microplastic life cycle and may have important ecological implications.