Road networks can affect human social connectivity and economic opportunities, as well as negatively impact freshwater ecosystems, including dependent species such as fishes and related ecosystem services. We summarize the negative impacts of roads on freshwater ecosystems and discuss how these interact with the biology of different fish species and change ecological communities. Poorly designed and constructed road infrastructure disrupts fishes’ daily movements and migration routes by altering geomorphology and hydrological connectivity. Freshwater ecosystems and fishes are negatively impacted by essentially all stages of a road’s lifespan, from the sedimentation caused by construction to the cumulative effects of flow regulation. The history of industrialization also means that much of the knowledge about how roads damage freshwater ecosystems comes from North America, Australia, and Europe, leaving gaps in knowledge for most of the world’s freshwater ecosystems and fish species. Cross-sectoral initiatives between government, environmental regulators, resource managers, academia (e.g., natural sciences, social sciences, and engineering), transportation agencies, forestry and agricultural producers, and other relevant groups are needed to better plan, regulate, and identify road infrastructure designs that minimize negative impacts on local and distant ecology.