The coexistence of large mammals and humans in the contemporary landscape is a big challenge for conservationists. Wild ungulates that forage on arable fields are exposed to the negative effects of pesticides, and this problem also applies to protected species for which intoxication by pesticides may pose a health risk and directly affect the effectiveness of conservation efforts. In this paper we assessed the threat posed by pesticides to the European bison Bison bonasus, a species successfully restituted after being extinct in the wild. We studied samples of B. bonasus liver from three free-living populations in Poland (Białowieska, Knyszyńska, and Borecka forests) and captive individuals from breeding centres. LC-QTOF-MS/MS two-step analysis for the detection, identification and confirmation of pesticide residues in liver samples, which included MS and targeted MS/MS scans, was conducted. It was found that European bison are exposed to pesticides as a result of crop depredation: the presence of tetraconazole, fluopyram and diazinon residues in 12 liver samples was confirmed. The concentration levels of the detected substances were quite low, but in the liver samples more than one substance was usually found, and the potential health risk to European bison may result from the synergistic interaction of these substances. The place of occurrence of the population, abundance, and the management regime affect the exposure of European bison to pesticides. Due to the high conservation status of the European bison, the monitoring of intoxication by pesticides should be included in the conservation plans of this species. This issue should also be more widely included in the study of other wild ungulates because knowledge about the impact of pesticides on wildlife is still insufficient.