Human-induced transformations lead to multiple threats to biodiversity in freshwater ecosystems mainly related to climate change, biological invasion, land use change, pollution, and overexploitation. These threats operate through multiple mechanisms, which can be complementary or compensatory. However, the effects of the major threats to biodiversity across ecological scales are still unclear, as well as its operating mechanisms. We performed a meta-analysis on their impacts on freshwater ecosystems to assess their general and relative importance across multiple ecological levels. We demonstrated that pollution was consistently the most important threat to freshwater ecosystems change, but the relative importance of each threat depended on the ecological level. At the population level, nutrient loading driven by pollution and climatic warming had higher relative importance, increasing metabolic rates through a bottom-up effect. However, this effect did not propagate to other ecological scales. Communities were more sensitive to the impacts of biological invasion and land use change, both synergically decreasing their diversity, evenness, and richness. At the ecosystem level, both pollution and land use change impacts were more relevant to eutrophication of freshwater ecosystems. We highlight the lack of information on impacts from overexploitation and studies demonstrating the combined effect among these major threats. We concluded that freshwater ecosystems are prone to these threats by a set of pathways in which their impacts are not equally widespread across the ecological levels, affecting them in a multidirectional way. We reinforce the importance of designing conservation strategies that allow counteracting the impacts of biodiversity loss by multiple pathways and including such multidirectionality to plan global actions to protect freshwater ecosystems.