Overexploitation, habitat fragmentation, and flow alteration are major threats to freshwater biodiversity that can lead to fisheries collapse and species extinction. These threats are particularly alarming in poorly monitored ecosystems where resource use supports the livelihoods of numerous people. The Tonle Sap Lake in Cambodia is such an ecosystem, supporting one of the world's largest freshwater fisheries. Tonle Sap Lake fishes are the focus of indiscriminate harvest affecting species stocks, community composition and food-web structure. Changes in the magnitude and timing of the seasonal flood pulse have also been linked to declines in fish stocks. Yet, changes in fish abundance and species-specific temporal trends remain poorly documented. Analyzing 17 years' time series of fish catch data for 110 species, we show that fish populations have declined by 87.7 %, owing to a statistically significant decline for >74 % species, particularly the largest ones. Despite large variations in species-specific trends - going from locally extinct to >1000 % increase - declines were found across most migratory behaviors, trophic positions or IUCN threat categories, though uncertainty regarding the magnitude of effect precluded us drawing conclusions in some cases. These results, reminiscent of alarming declines in fish stocks in many marine fisheries, provide unequivocal evidence that Tonle Sap fish stocks are increasingly depleted. The consequences of this depletion on ecosystem function are unknown but will undoubtedly affect the livelihoods of millions of people, stressing the need to set-up management strategies aimed to protect both the fishery and its associated diversity. Flow alteration, habitat degradation / fragmentation - especially deforestation of seasonally inundated areas and overharvest - have been reported as major drivers in population dynamics and community structure, highlighting the need for management efforts aimed at preserving the natural flood pulse, protecting flooded forest habitats, and reducing overfishing.