Microplastics (MP) are particles under 5 mm that are widespread in the environment and can harm aquatic life by accumulating pollutants. Understanding their distribution and the effects of urbanization and seasonality is key to assessing their impact and developing mitigation strategies. This study investigated the influence of these factors on MP profiles and associated risks in the São Bartolomeu Stream in Viçosa (Minas Gerais, Brazil). Water samples were collected upstream and downstream of the urban center during dry and rainy seasons. Urbanization and seasonality were found to significantly impact MP presence. The abundance of suspected MP particles increased downstream of the urban center during the dry season, from 1.7 to 2.6 MP particles per liter (MP L⁻¹), whereas no significant difference was observed during the rainy season. Subsequent MP identification using Raman spectroscopy confirmed polymer concentrations ranging from 0.05 to 0.32 MP L⁻¹, with a predominance of polypropylene (PP) and polyethylene (PE). However, a critical paradox emerged: standard ecotoxicity tests with the most abundant polymers (PE and PP) revealed no acute or chronic effects on Daphnia similis. However, a risk assessment driven by the chemical identity of less abundant but highly hazardous polymers found in the stream (including polyvinyl chloride, polyacrylonitrile, and polyurethane), classified the aquatic environment with a ‘high’ to ‘extremely high’ ecological risk, highlighting a potential threat from the mixed polymer composition. Thus, the present study reinforces the need for continuous monitoring and mitigation measures to reduce MP contamination in urban aquatic environments.