Simple SummaryThe wild boar (Sus scrofa) has been identified as a reservoir of zoonoses and food-borne pathogens. Wild boar populations are growing worldwide, also near urban centers such as Barcelona (Catalonia, Spain). The aim of this study was to assess the presence of zoonotic agents in the wild boar populations from the Metropolitan Area of Barcelona (MAB). The detection of Escherichia coli carrying critical antimicrobial resistance genes to β-lactams and, for the first time in Spain, the detection of toxigenic Clostridioides difficile strains in wild boars foraging in urban areas show the value of this game species as a sentinel of antimicrobial-resistant bacteria (AMRB) and zoonotic agents in the environment. Moreover, the wild boars foraging in urban and peri-urban locations were more exposed to AMRB sources than the wild boars dwelling in natural environments. The responsible health agencies should undertake specific actions to fully assess the potential implications for the human population and/or environmental health. Zoonotic diseases and hazards beyond the compulsory surveillance diseases should be specifically targeted in the growing human–wildlife interface of urban environments such as the MAB under the One Health approach.Disease transmission among wild boars, domestic animals and humans is a public health concern, especially in areas with high wild boar densities. In this study, fecal samples of wild boars (n = 200) from different locations of the Metropolitan Area of Barcelona were analyzed by PCR to explore the frequency of β-lactamases and extended cephalosporin and carbapenem resistance genes (ESBLs) in Escherichia coli strains and the presence of toxigenic Clostridioides difficile. The prevalence of genes conferring resistance to β-lactam antimicrobials was 8.0% (16/200): blaCMY-2 (3.0%), blaTEM-1b (2.5%), blaCTX-M-14 (1.0%), blaSHV-28 (1.0%), blaCTX-M-15 (0.5%) and blaCMY-1 (0.5%). Clostridioides difficile TcdA+ was detected in two wild boars (1.0%), which is the first report of this pathogen in wild boars in Spain. Moreover, the wild boars foraging in urban and peri-urban locations were more exposed to AMRB sources than the wild boars dwelling in natural environments. In conclusion, the detection of E. coli carrying ESBL/AmpC genes and toxigenic C. difficile in wild boars foraging in urban areas reinforces the value of this game species as a sentinel of environmental AMRB sources. In addition, these wild boars can be a public and environmental health concern by disseminating AMRB and other zoonotic agents. Although this study provides the first hints of the potential anthropogenic sources of AMR, further efforts should be conducted to identify and control them.