Antimicrobial resistance (AMR) is a burgeoning environmental concern demanding a comprehensive One Health investigation to thwart its transmission to animals and humans, ensuring food safety. Seafood, housing bacterial AMR, poses a direct threat to consumer health, amplifying the risk of hospitalization, invasive infections, and death due to compromised antimicrobial treatments. The associated antimicrobial resistance genes (ARGs) in diverse marine species can amass and transmit through various pathways, including surface contact, respiration, and feeding within food webs. Our research, focused on the English Channel and North Sea, pivotal economic areas, specifically explores the occurrence of four proposed AMR indicator genes (tet(A), blaTEM, sul1, and intI1) in a benthic food web. Analyzing 350 flatfish samples' skin, gills, and gut, our quantitative PCR (qPCR) results disclosed an overall prevalence of 71.4% for AMR indicator genes. Notably, sul1 and intI1 genes exhibited higher detection in fish skin, reaching a prevalence of 47.5%, compared to gills and gut samples. Proximity to major European ports (Le Havre, Dunkirk, Rotterdam) correlated with increased AMR gene frequencies in fish, suggesting these ports' potential role in AMR spread in marine environments. We observed a broad dispersion of indicator genes in the English Channel and the North Sea, influenced by sea currents, maritime traffic, and flatfish movements. In conclusion, sul1 and intI1 genes emerge as robust indicators of AMR contamination in the marine environment, evident in seawater and species representing a benthic food web. Further studies are imperative to delineate marine species' role in accumulating and transmitting AMR to humans via seafood consumption. This research sheds light on the urgent need for a concerted effort in comprehending and mitigating AMR risks in marine ecosystems within the context of One Health.