Abstract: As hydropower development expands across lowland tropical forests, flooding and concomitant insular fragmentation have become important threats to biodiversity. Newly created insular landscapes serve as natural laboratories to investigate biodiversity responses to fragmentation. One of these most iconic landscapes is the Balbina Hydroelectric Reservoir in Brazilian Amazonia, occupying >400 000 ha and comprising >3 500 forest islands. Here, we synthesise the current knowledge on responses of a wide range of biological groups to insular fragmentation at Balbina. Sampling has largely concentrated on a set of 22 islands and three mainland sites. In total, 39 studies were conducted over nearly two decades, covering 17 vertebrate, invertebrate, and plant taxa. Although species responses varied according to taxonomic group, island area was consistently included and played a pivotal role in 66.7% of all studies examining patterns of species diversity. Species persistence was further affected by species traits, mostly related to species capacity to use/traverse the aquatic matrix or tolerate habitat degradation, as noted for species of vertebrates and orchid bees. Further research is needed to improve our understanding of such effects on wider ecosystem functioning. Environmental Impact Assessments must account for changes in both the remaining habitat amount and configuration, and subsequent long-term species losses.