The Pinheiros River in São Paulo, Brazil, crosses through the capital city and has its confluence with the River Tiete, which comprises several reservoirs along its course. Although Pinheiros River is considered one of the heaviest polluted rivers in Brazil, little is known about its bacterial composition, their metabolic functions or how these communities are affected by the physicochemical parameters of the river. In this study, we used the 16S rRNA gene Illumina MiSeq sequencing to profile the bacterial community from the water surface at 11 points along the course of the River. Taxonomical composition revealed an abundance of Proteobacteria phyla, followed by Firmicutes and Bacteroidetes, with a total of 233 classified bacterial families and 558 known bacterial genera. Among the 35 potentially pathogenic bacteria identified, Arcobacter was the most predominant genus. The disrupted physicochemical parameters detected in this study may possibly contribute to the composition and distribution of the bacterial community in the Pinheiros River. Predictive functional analysis suggests the River is abundant in motility genes, including bacterial chemotaxis and flagellar assembly. These results provide novel and detailed insights into the bacterial communities and putative function of the surface water in the Pinheiros River.