The Pinheiros River of São Paulo, the southeastern state in 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 and their metabolic functions and how these communities are affected by the physical-chemical parameters of the river. In this study, we used the 16S rRNA gene Illumina MiSeq sequencing to profile the structure of the bacterial community from the water surface of 11 points along the course of the River. Taxonomical composition revealed the abundance of Proteobacteria phyla, followed by Firmicutes and Bacteroidetes, with a total of 233 classified bacterial families and 558 known bacterial genera.