Background: Upwelling systems are characterised by an intense primary biomass production in the surface (warmest) water after the outcrop of the bottom (coldest) water, which is rich in nutrients. Although it is known that the microbial assemblage plays an important role in the food chain of marine systems and that the upwelling systems that occur in southwest Brazil drive the complex dynamics of the food chain, little is known about the microbial composition present in this region. Methodology/Principal Findings: We carried out a molecular survey based on SSU rRNA gene from the three domains of the phylogenetic tree of life present in a tropical upwelling region (Arraial do Cabo, Rio de Janeiro, Brazil). The aim was to analyse the horizontal and vertical variations of the microbial composition in two geographically close areas influenced by anthropogenic activity (sewage disposal/port activity) and upwelling phenomena, respectively. A lower estimated diversity of microorganisms of the three domains of the phylogenetic tree of life was found in the water of the area influenced by anthropogenic activity compared to the area influenced by upwelling phenomena. We observed a heterogenic distribution of the relative abundance of taxonomic groups, especially in the Archaea and Eukarya domains. The bacterial community was dominated by Proteobacteria, Cyanobacteria and Bacteroidetes phyla, whereas the microeukaryotic community was dominated by Metazoa, Fungi, Alveolata and Stramenopile. The estimated archaeal diversity was the lowest of the three domains and was dominated by uncharacterised marine Crenarchaeota that were most closely related to Marine Group I. Conclusions/Significance: The variety of conditions and the presence of different microbial assemblages indicated that the area of Arraial do Cabo can be used as a model for detailed studies that contemplate the correlation between pollutionindicating parameters and the depletion of microbial diversity in areas close to anthropogenic activity; functional roles and geochemical processes; phylogeny of the uncharacterised diversity; and seasonal variations of the microbial assemblages. © 2011 Cury et al.