Extreme weather events and the presence of mega-hydroelectric dams, when combined, present an emerging threat to natural habitats in the Amazon region. To understand the magnitude of these impacts, we used remote sensing data to assess forest loss in areas affected by the extreme 2014 flood in the entire Madeira River basin, the location of two mega-dams. In addition, forest plots (26 ha) were monitored between 2011 and 2015 (14,328 trees) in order to evaluate changes in tree mortality, aboveground biomass (AGB), species composition and community structure around the Jirau reservoir (distance between plots varies from 1 to 80 km). We showed that the mega-dams were the main driver of tree mortality in Madeira basin forests after the 2014 extreme flood. Forest loss in the areas surrounding the reservoirs was 56 km² in Santo Antônio, 190 km² in Jirau (7.4–9.2% of the forest cover before flooding), and 79.9% above that predicted in environmental impact assessments. We also show that climatic anomalies, albeit with much smaller impact than that created by the mega-dams, resulted in forest loss along different Madeira sub-basins not affected by dams (34–173 km²; 0.5–1.7%). The impact of flooding was greater in várzea and transitional forests, resulting in high rates of tree mortality (88–100%), AGB decrease (89–100%), and reduction of species richness (78–100%). Conversely, campinarana forests were more flood-tolerant with a slight decrease in species richness (6%) and similar AGB after flooding. Taking together satellite and field measurements, we estimate that the 2014 flood event in the Madeira basin resulted in 8.81–12.47 ∙ 10⁶ tons of dead biomass. Environmental impact studies required for environmental licensing of mega-dams by governmental agencies should consider the increasing trend of climatic anomalies and the high vulnerability of different habitats to minimize the serious impacts of dams on Amazonian biodiversity and carbon stocks.