Freshwater ecosystems cover over 15% of the world’s surface and provide ecosystem services that are pivotal in sustaining human society. However, fast-growing anthropogenic activities have deleterious impacts on these ecosystems. In this Special Issue, we collect ten studies encompassing five different factors of freshwater contamination: landfill leaks, nutrients, heavy metals, emerging organic contaminants and marble slurry. Using different approaches, the studies detailed the direct and indirect effects that these contaminants have on a range of freshwater organisms, from bacteria to vertebrates. Although the papers covered here focused on specific case studies, they exemplify common issues that are expanding in groundwaters, hyporheic zones, streams, lakes and ponds around the world. All the aspects of these issues are in dire need of being continuously discussed among scientists, end-users and policy-makers. To this end, the Special Issue presents a new free software suite for the analysis of the ecological risk and conservation priority of freshwater ecosystems. The software can support local authorities in the preparation of management plans for freshwater basins pursuant to the Water Directives in Europe.