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Abstract Background Although antibiotics have significantly improved human and animal health, their intensive use leads to the accumulation of antimicrobial resistance (AMR) in the environment. Moreover, certain waste management practices create the ideal conditions for AMR development while providing predictable resources for wildlife. Here, we investigated the role of landfills in the potentiation of New World vultures to disseminate environmental AMR. We collected 107 samples (soil, water, and feces) between 2023 and 2024, in different bird use sites (roosts, landfills and boneyards). Results We isolated enterococci (EN), Escherichia coli (EC), and Salmonella spp. (SM), performed antibiotic susceptibility tests, and quantified the presence of antibiotic resistance genes (ARGs) within all samples. We identified EN, EC, and SM, in 50, 37, and 26 samples, from the three vulture use areas, respectively. AMR was mainly to aminoglycoside, cephalosporin, and tetracycline, and the prevalence of multidrug resistance (MDR) was 5.3% (EC), 78.2% (EN), and 17.6% (SM). Variations in bacterial abundance and AMR/MDR profiles were found based on the season, use site, and sample types, which was corroborated by ARG analyses. Conclusions Our study suggests that landfills constitute a source of zoonotic pathogens and AMR for wildlife, due to readily available refuse input. Using non-invasive molecular methods, we highlight an often-ignored ecosystem within the One Health paradigm.