Globally, urbanization continues to increase and produce elevated levels of contaminants (e.g., metals, pesticides) in the air, soil, and water within and around urban centers. In particular, heavily trafficked roads are a major source of anthropogenic contaminants, including polycyclic aromatic hydrocarbons (PAHs), heavy metals, tire dust, and road salt. This mixture, herein referred to as road runoff, represents one of the largest contributors of diffuse-source toxicants in urban areas, and yet is seldom studied as a contaminant mixture. Here we investigate how road runoff could impact ecosystem structure and function in urban stream ecosystems. Specifically, we (1) investigate contaminant fluctuations over winter, a time of intense road salt application, (2) assess community-level invertebrate responses to contamination loads in urban streams, and (3) assess the effect of road runoff-associated chemicals to early life stage freshwater mussels. Preliminary results demonstrate that contaminant concentrations (chloride, metals, microplastics) in urban sites are greater than rural sites. Additionally, the amount of chloride has a strong relationship with temperature, where chloride concentrations increase with air temperature during the winter season. Last, we detected differences in community composition of benthic macroinvertebrates between rural and urban sites. Future work will determine how early life stage freshwater mussels respond to environmentally relevant concentrations of each road runoff contaminant both independently and combined in a full factorial experiment. This will provide insight into potential stressors that can impact these and other sensitive species. This study builds on our understanding of how multiple anthropogenic stressors impact stream communities, and has the potential to help inform future policy and urban development decisions.