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Background: Planktonic bacteria are recognized as important drivers of biogeochemical processes in all aquatic ecosystems, however, the taxa that make up these communities are poorly known. The aim of this study was to investigate bacterial communities in aquatic ecosystems at Ilha Grande, Rio de Janeiro, Brazil, a preserved insular environment of the Atlantic rain forest and how they correlate with a salinity gradient going from terrestrial aquatic habitats to the coastal Atlantic Ocean. Methodology/Principal Findings: We analyzed chemical and microbiological parameters of water samples and constructed 16S rRNA gene libraries of free living bacteria obtained at three marine (two coastal and one offshore) and three freshwater (water spring, river, and mangrove) environments. A total of 836 sequences were analyzed by MOTHUR, yielding 269 freshwater and 219 marine operational taxonomic units (OTUs) grouped at 97% stringency. Richness and diversity indexes indicated that freshwater environments were the most diverse, especially the water spring. The main bacterial group in freshwater environments was Betaproteobacteria (43.5%), whereas Cyanobacteria (30.5%), Alphaproteobacteria (25.5%), and Gammaproteobacteria (26.3%) dominated the marine ones. Venn diagram showed no overlap between marine and freshwater OTUs at 97% stringency. LIBSHUFF statistics and PCA analysis revealed marked differences between the freshwater and marine libraries suggesting the importance of salinity as a driver of community composition in this habitat. The phylogenetic analysis of marine and freshwater libraries showed that the differences in community composition are consistent. Conclusions/Significance: Our data supports the notion that a divergent evolutionary scenario is driving community composition in the studied habitats. This work also improves the comprehension of microbial community dynamics in tropical waters and how they are structured in relation to physicochemical parameters. Furthermore, this paper reveals for the first time the pristine bacterioplankton communities in a tropical island at the South Atlantic Ocean. [ABSTRACT FROM AUTHOR]