The river Nile flows across 11 African countries, supporting millions of human livelihoods, and holding globally important biodiversity and endemism yet remains underprotected. No basin-wide spatial conservation planning has been attempted to date, and the importance of coordinated conservation planning for the Nile's biodiversity remains unknown. We address these gaps by creating a basin-wide conservation plan for the Nile's freshwater fish. We identify priority areas for conservation action and compare cross-boundary collaboration scenarios for achieving biodiversity conservation targets, accounting for river connectivity. We found that collaborative conservation efforts are crucial for reducing conservation costs, saving 34% of costs compared to an uncoordinated, business-as-usual scenario. While most Nile basin countries benefit from coordinating conservation planning, costs and benefits are unequally distributed. We identify "hot spots" consistently selected as conservation priority areas across all collaboration scenarios, and provide a framework for improving return on conservation investment for large and complex river systems globally.