Biological corridors are connections which link habitats in a regional scale, allowing the gene flow between populations. The Uruguay River comprises riverside and insular riparian forests along subtropical to temperate zones passing through different biogeographic provinces. The aim of this study was to characterise the spider fauna from the Uruguay River islands highlighting their connection role for the spider community of riparian forest. Spiders were studied from surveys in a fluvial island of the southern course of the river with nine campaigns being carried out from September 2007 to September 2009. Three complementary collecting methods were used: G-Vac, night hand collecting and pitfall traps. A total of 58 samples were taken in each campaign. A total of 33 families, 145 species/morphospecies and 8 guilds were registered. Theridiidae and space web weavers showed the highest abundance and species richness. Web weavers were predominant in the spider community evidencing the importance of the forest vegetation heterogeneity in spider diversity. Fifteen species have been recorded for the first time for Uruguay. Additional data of previous surveys in the northern islands of the river were analysed and compared. Several species confirm the role of the Uruguay River as a biological corridor from the upper to lower course of the river. The riparian forests from the islands constitute a southernmost intromission of Paranaense biota between Chaco and Pampa regions along the river. The results obtained are an important input for the conservation of these areas. Knowing the biodiversity, as well as its dynamics and the flow of biota that exists in these environments, would allow planning the management from a regional point of view.