Threatened by multiple stressors including habitat loss and fragmentation, climate change, invasive species, and water pollution and abstraction by humans, freshwater biodiversity is the fastest declining part of the global biota. A multitude of recent agenda-setting publications have pointed out key objectives and goals for addressing this freshwater biodiversity crisis, but important gaps must be overcome to reach ambitious conservation targets. Meanwhile, the Kunming-Montreal post-2020 Global Biodiversity Framework (GBF) calls for transformative change in worldwide approaches to biodiversity conservation, setting an ambitious plan for coming decades. In this perspective, we synthesize recent literature in freshwater biodiversity conservation with major topics in the GBF by highlighting important gaps in knowledge, governance and legislation, and implementation. This gaps-oriented approach is designed to facilitate meaningful action by highlighting missing “pieces” in the conservation process that must be addressed to achieve the ambitious goals set for global conservation. We derive 14 overarching gaps from a conference session and informal synthesis of recent literature in freshwater biodiversity conservation to catalyze research, advocacy, and action toward these goals. Key gaps include inventory data on global freshwater biodiversity, collating and mobilizing conservation evidence in practice, improving coordination of ecological governance at scale, including within and across catchments, and navigating trade-offs between economic development, resource consumption, and priorities for freshwater biodiversity. Finally, we apply this gaps-oriented approach to key language describing GBF goals for freshwater biodiversity conservation, and point out existing and emerging solutions which may help address important gaps. Major themes that address multiple gaps include the use of Nature-based Solutions and Other Effective Area-based Conservation mechanisms (OECMs), navigation of water management trade-offs with Indigenous and local people and other stakeholders, and funding and policy mechanisms to facilitate conservation action and support meaningful monitoring of conservation evidence at the watershed scale.