Soybean has undergone the greatest expansion of any global crop, fuelled by the emergence of herbicide-resistant crops. In Uruguay, soybean croplands have increased from virtually zero to more than 1 million ha in 20 years. Uruguay is also implementing its system of protected areas. Here, we assess the presence of pesticides within a Ramsar site and protected area, in a basin dominated by croplands. We consider pesticides as surrogates of the subtle impacts of agribusiness on conservation initiatives and other productions. Pesticides were found in soils, fishes and beehives, both within and around the protected area. Endosulfan was found in all matrices analysed (23 of 80 samples), while glyphosate (0–2.31 mg/kg) and aminomethylphosphonic acid (AMPA; 0–0.61 mg/kg) were found in all soil classes. The study also allowed for a retrospective evaluation of a recent policy banning endosulfan in Uruguay, suggesting that while the protected area has not been immune to the impacts of agribusiness on human health or biodiversity, limiting the use of pesticides reduces or avoids some of them. This has implications for the design of multifunctional landscapes and for the debate on land sharing versus land sparing.