One of the biggest environmental problems generated by anthropocentric activities is the pollution of water sources. Monitoring these areas is of great importance for maintaining their quality and for human heath, because the distribution of drinking water depends on the river ecosystem. This study aimed to realize the biomonitoring, at different trophic levels, of the Marrecas River Waters (Paraná, Brazil). The samples were collected in 2020 at nine points, three in the rural area and six in the urban area along the Marrecas River. Physicochemical and microbiological analyses, toxicity test with Artemia salina, escape test with Eisenia fetida, cytotoxicity with mammalian cells and cytotoxicity and mutagenicity test with Allium cepa were realized. The results show that the Marrecas River water quality is compromised, mainly in the urban region, possibly due to the inadequate use and occupation of its basin and inadequate disposal of effluents in the region. In addition, the study shows the importance of testing at different trophic levels, as they have different sensitivity, as is the case of A. cepa, which was the only one to demonstrate high levels of mutagenicity for the source of the Marrecas River.