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Home to one of the world's most biodiverse and productive freshwater fisheries, the Mekong River basin provides food and economic security to some 60 million people. The extensive hydropower development planned for the river basin has the potential to degrade this ecosystem. The basin's potentially most impactful and downstream-most proposed main stem dam, the 18-km-long Sambor Dam is to be located just upstream of critical nursery and fishery habitats of the Tonle Sap Lake and the Mekong Delta and just downstream of major spawning areas. We explore the potential to modify Sambor's siting, design, and operation (SDO) to balance hydropower and ecological outcomes. Our results demonstrate that there are opportunities to achieve more balanced ecology–energy outcomes. We use probabilistic seasonal measures of fish life cycle processes, energy production, and sediment passage to explore alternative SDO options. We consider both ecological concerns and energy production on equal footing prior to any power purchase agreements or fixed designs for Sambor, which is a first among any of the world's large dams. The Sambor Ecological Alternative (Sambor EA) dam considered in this study includes a combination of design and operational features to improve sediment and fish passage that do not exist at any of the world's large hydropower dams. Yet Sambor EA still poses a significant risk to the Mekong fishery and the millions who depend on it, given the appreciable uncertainty surrounding its potential ecological outcomes. Our results emphasize the need to address ecological concerns in advance of signing power purchase agreements that would otherwise limit the site-specific and integrated SDO modifications needed to achieve them. Our results also emphasize the importance of, and challenges in, carefully defining ecological performance measures and criteria, as well as the importance of carefully sampling reservoir operation possibilities. [ABSTRACT FROM AUTHOR]