Microplastic pollution is an urgent threat to the biota of aquatic ecosystems and is generally recognized as a global issue. Identifying the sources of microplastics is acknowledged as the most effective approach for mitigating microplastic pollution. However, the factors that regulate the spatiotemporal dynamics of microplastics in urban river networks, such as microplastic sources and other variables, have not been studied together at the watershed scale, let alone regarding their impact on internal microplastics. Here, we define "internal microplastics" as microplastics in biota, either in the digestive system or internal organs of organisms. We estimated the effects of anthropogenic activities (land cover and wastewater treatment plants) and seasonality on the concentration of internal microplastics in midge larvae (Diptera: Chironomidae) in an urban river system at a watershed scale in Taiwan. Agricultural activities, but not industrial activities, had a significant negative nonlinear effect on microplastic concentration. However, seasonality was the most crucial factor, as the microplastic concentration was significantly lower during the wet season. Although the presence of a wastewater treatment plant significantly increased the microplastic concentration at downstream sampling sites, its effect appeared to be minor. We conclude that seasonality overrides the effects of anthropogenic activities on the variation in the concentration of internal microplastics in midge larvae in an urban river system.