<p>Although agrochemicals protect crops and reduce losses, these chemicals can migrate to non-target environments via run-off and leaching following irrigation or heavy rainfall, where non-target organisms can be exposed to a mixture of water-soluble compounds. This study investigated whether the water-soluble fractions of selected agricultural soils from South Africa contain quantifiable concentrations of four commonly used pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D), atrazine, dicamba and imidacloprid, and whether the aqueous extracts induce effects in vitro. Effects investigated included cytotoxicity using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell viability assay; xenobiotic metabolism using the H4IIE-<i>luc</i> rat hepatoma cell line; and (anti-)androgenic and (anti-)oestrogenic effects were screened for with the human breast carcinoma cell lines MDA-kb2 and T47D-KB<i>luc</i>, respectively. Oxidative stress responses were measured in H4IIE-<i>luc</i> and human duodenum adenocarcinoma (HuTu-80) cells. All extracts of soil induced oxidative stress, while several samples caused moderate to severe cytotoxicity and/or anti-androgenic effects<i>.</i> The herbicide atrazine had the greatest frequency of detection (89%), followed by dicamba (84%), 2,4-D (74%) and imidacloprid (32%). Concentrations of atrazine [2.0 × 10^–1 to 2.1 × 10²&#xa0;ng/g, dry mass (dm)] and the neonicotinoid insecticide, imidacloprid (2.0 × 10¹ to 9.7 × 10¹&#xa0;ng/g, dm), exceeded international soil quality guidelines. Overall, there was no observable trend between the biological effects and pesticides quantified. Nonetheless, the findings of this study show that agricultural soils in South Africa can elicit effects in vitro and contain quantifiable concentrations of polar pesticides. These agrochemicals might pose risks to the health of humans and the environment, but more assessment is necessary to quantify such potential effects.</p>