The Tapajós is a main tributary to the Amazon River, harboring a large biological and cultural diversity. However, despite its socioecological importance, the Tapajós experiences multiple anthropogenic pressures arising from deforestation and forest fires, illegal artisanal gold mining, and agribusiness and infrastructure development. As part of the Águas do Tapajós (Water of Tapajós) project, an integrated diagnosis of water quality and biodiversity, as well as of artisanal fishing and subsistence agriculture practiced in eight communities located along the Lower and Middle Tapajós was conducted. Results indicated a decrease in water transparency from 54 to 73% of the levels observed in a study from 1982, probably associated with the input of sediments from deforestation and mining. These activities were also potentially related to the high levels of mercury observed in the riverine population sampled, in average 4.6 times higher than the limit recommended by the World Health Organization (WHO). Furthermore, the presence of microcystin, a toxin that can cause liver failure and death, was detected in the vicinity of seven communities. In one of the sampled points, the level measured was higher than WHO’s recommended limit for drinking water. Microplastic waste was recorded at all collection stations and sampling periods. High densities of fish eggs and larvae deriving provided evidence that the Tapajós River is an important reproduction site for ichthyofauna. This can be reinforced by the large number (326) of species of fish reported, 190 of which with recognized potential for use in commercial and subsistence fishing. Capture of chelonians, although illegal, occurred in all the communities studied, either for consumption or selling, and a decline in the abondance of turtles was reported. As expected, fish was declared the main source of protein for the families interviewed, 86% of which, in addition to fishing, practice subsistence agriculture. However, there was a general perception of decline in the abondance of 31 species of fish. Overall, the study indicated a tendency towards deterioration of water quality conditions and aquatic biodiversity in the Tapajós River, particularly fishing resources. The worsening conditions can affect the health and water and food security of riverine communities. This scenario reinforces the importance of continued monitoring, as well as effective actions to contain the root causes of the observed degradation.