We describe the composition of bacterial assemblages in different localities from the Rimac river basin, the main source of drinking water for Lima, the capital of Peru, and analyze the occurrence of pathogenic bacteria that are nor usually reported in traditional microbiological assessments. The dominance of pathogens, that are not usually detected in surveys of water microbiological quality performed by the Peruvian government, highlights the necessity to apply metabarcoding tools for surveillance of emergent and unculturable bacteria. This is the first report of the diversity of bacterial assemblages from the Rimac river using a metabarcoding approach.