Seafood and the marine environment are often considered as potential reservoirs of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs); however, there are few studies and sparse results on this sector. This study aimed at providing new data and insights regarding the content of resistance markers in various seafood samples and sources, and therefore the potential exposure to humans in a global One Health approach. An innovative high throughput qPCR screening was developed and validated in order to simultaneously investigate the presence of 41 ARGs and 33 MGEs including plasmid replicons, integrons, and insertion sequences associated to Gram-negative bacteria. Analysis of 268 seafood isolates from the bacterial microflora of cod (n = 24), shellfish (n = 66), flat fishes (n = 53), shrimp (n = 10), and horse mackerel (n = 115) showed the occurrence of sul-1, ant(3”)-Ia, aph(3’)-Ia, strA, strB, dfrA1, qnrA, and blaCTX-M-9-group genes in Pseudomonas spp., Providencia spp., Klebsiella spp., Proteus spp., and Shewanella spp. isolates. MGEs were identified in all bacterial species investigated. We found that the occurrence of MGE may be associated with the seafood type and the environmental, farming, and harvest conditions. Moreover, even if MGE were detected in half of the seafood isolates investigated, association with ARG was only identified for twelve isolates. Our results corroborate the hypothesis that the incidence of antimicrobial-resistant bacteria and ARGs decreases with increasing distance from potential sources of fecal contamination. Moreover, we provide unique and original high throughput micro-array designed for the screening of ARGs and MGE in Gram-negative bacteria, which is easily implementable for monitoring antimicrobial resistance gene markers in diverse contexts.