<p>The consumption of freshwater from wells and springs is common on small properties in southern Brazil and often takes place in areas used for crop cultivation. In this context, this study aimed to investigate the ecotoxicity of water resources and adjacent soils in three agricultural regions in southern Brazil (Paraná-PR, Santa Catarina-SC, and Rio Grande do Sul-RS). Water and soil samples were collected four times over the course of 1&#xa0;year. Ecotoxicological monitoring was conducted using cytotoxicity and mutagenicity tests on <i>Allium cepa</i> and the bioluminescent bacterium <i>Vibrio fischeri</i> (Microtox System). The <i>A. cepa</i> tests showed that soils adjacent to water resources in PR (winter) and SC (summer) exhibited cytotoxicity, which in the case of PR was associated with high iron concentrations in the soil. Water samples did not show cytotoxic or mutagenic effects in any of the <i>A. cepa</i> assays. In the <i>V. fischeri</i> tests, the highest toxicity was observed in the water sample from PR (spring), with an effective concentration (EC₅₀) value of 15.51%. Multivariate analysis revealed changes in the quality of the water sources. This type of study is important for assessing the environmental quality of natural water resources under the influence of anthropogenic activities, enabling the evaluation and implementation of measures to protect freshwater quality.</p>