The Negro River is located in the Amazon basin, the largest hydrological catchment in the world. Its water is used for drinking, domestic activities, recreation and transportation and water quality is significantly affected by anthropogenic impacts. The goals of this study were to determine the presence and concentrations of the main viral etiological agents of acute gastroenteritis, such as group A rotavirus (RVA) and genogroup II norovirus (NoV GII), and to assess the use of human adenovirus (HAdV) and JC polyomavirus (JCPyV) as viral indicators of human faecal contamination in the aquatic environment of Manaus under different hydrological scenarios. Water samples were collected along Negro River and in small streams known as igarape´s. Viruses were concentrated by an organic flocculation method and detected by quantitative PCR. From 272 samples analysed, HAdV was detected in 91.9 %, followed by JCPyV (69.5 %), RVA (23.9 %) and NoV GII (7.4 %). Viral concentrations ranged from 102 to 106 GC L-1 and viruses were more likely to be detected during the flood season, with the exception of NoV GII, which was detected only during the dry season. Statistically significant differences on virus concentrations between dry and flood seasons were observed only for RVA. The HAdV data provides a useful complement to faecal indicator bacteria in the monitoring of aquatic environments. Overall results demonstrated that the hydrological cycle of the Negro River in the Amazon Basin affects the dynamics of viruses in aquatic environments and, consequently, the exposure of citizens to these waterborne pathogens.