Pollution of water bodies can cause environmental and public health problems. The Cavouco stream is a tributary of the Capibaribe River, one of the main rivers in the state of Pernambuco, Brazil, and receives a high pollution load from residential, laboratory and hospital effluents. The aim of the present study was to perform phenotypic and molecular characterization in this stream, and evaluate the water quality using microbiological parameters. Water was collected from five sampling points, and bacterial species were identified using biochemical and molecular methods through 16S rRNA gene sequence analysis. Total and thermotolerant coliforms were also quantified. Fermenting Gram-negative bacilli from the family Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis), non-fermenting bacilli (Pseudomonas aeruginosa and Pseudomonas putida) and Gram-positive bacilli (Bacillus cereus, Bacillus licheniformis, Bacillus pumilus and Staphylococcus hominis) were identified. A total of 25 bacterial isolates were phenotypically identified. All phenotypic identifications were confirmed by molecular analysis, except for S. hominis, which was molecularly identified as Exiguobacterium. Regarding water quality, all analyzed samples were positive for total and thermotolerant coliforms. The results obtained suggest that the Cavouco stream presents a potential risk for transmission of water-borne diseases, because of the presence of pathogenic bacteria. In addition, the current state of the stream also threatens the conservation of its native species.