The principles of island biogeography are rarely used to investigate the animal assemblages of Amazonian river islands. Here, we apply this approach to compare bird assemblages of Amazonian river islands with a variety of mainland habitats. We also examine how bird species diversity and composition are related to island physical attributes. Birds were sampled with mist nets and qualitative censuses on 11 river islands and at 24 mainland sites on the lower reaches of the Rio Negro in the Brazilian Amazon. Island bird assemblages are characterized by lower species richness and a higher abundance of a few dominant species. Additionally, species composition of the islands was significantly distinct from the mainland, including the nearby floodplain habitats. Number of bird species increased with island size and habitat diversity, and decreased with degree of isolation. In addition, small islands tended to harbour an impoverished subset of the species present on larger ones. Bird species diversity and composition on Amazonian river islands are likely influenced by the ecological succession and historical events affecting island formation. Considering their small total area across the Amazon basin, these insular fluvial environments could be disproportionally threatened by river channel disturbances such as climate change events, and hydroelectric building.