The widespread and excessive use of pesticides in modern agricultural practices has caused pesticide contamination of the environment, animals, and humans, with confirmed serious health consequences. This study aimed to identify the 20 most critical substances based on an analysis of detection frequency (DF) and median concentrations (MC) across environmental and biological matrices. A sampling campaign was conducted across 10 case study sites in Europe and 1 in Argentina, each encompassing conventional and organic farming systems. We analysed 209 active substances in a total of 4609 samples. All substances ranked among the 20 most critical were detected in silicon wristbands worn by humans and animals and indoor dust from both farming systems. Five of them were detected in all environmental matrices. Overall, higher values of DF and MC, including in the blood plasma of animals and humans, were recorded in samples of conventional compared to organic farms. The differences between farming systems were greater in the environmental samples and less in animal and human samples. Ten substances were detected in animal blood plasma from conventional farms and eight in animal blood plasma from organic farms. Two of those, detected in both farming systems, are classified as hazardous for mammals (acute). Five substances detected in animal blood plasma from organic farms and seven detected in animal blood plasma from conventional farms are classified as hazardous for mammals (dietary). Three substances detected in human blood plasma are classified as carcinogens. Seven of the substances detected in human blood plasma are classified as endocrine disruptors. Six substances, of which five were detected in human blood plasma, are hazardous for reproduction/development. Efforts are needed to elucidate the unknown effects of mixtures, and it is crucial that such research also considers biocides and banned substances, which constitute a baseline of contamination that adds to the effect of substances used in agriculture.< Réduire