Marine and ocean environments are the most widespread habitats in the world but are still among the least studied from the aspect of antibiotic resistance (AR). The aim was to investigate the seasonal and spatial features of AR in microbial communities along trophic gradient of central Adriatic encompassing a 68-km transect from wastewater-impacted estuary to coastal and pristine open sea. Eighty microbiomes were profiled by using Illumina-based 16S rRNA amplicon sequencing revealing that biogeographical shifts in microbial community are largely driven by the seasonality, featured by thermohaline stratification and nutrients in particular. Proteobacteria were the predominant along the gradient and major contributors to the marine resistomes. Targeted resistance genes were quantified using RT-qPCR, among which distribution of tetA, blaCTX-M and blaTEM correlated with higher temperature, nutrient load and lower salinity. WHO 'high' and 'critical priority' pathogens including vancomycin-resistant Enterococcus faecium and carbapenemase-producing Enterobacteriaceae were isolated from wastewater-impacted coastal waters, pointing to their sewage-driven dissemination in marine environment. Enterobacteriaceae carried carbapenemase genes (blaKPC-2, blaOXA-48 and blaVIM- 1) on diverse conjugative plasmids favouring their horizontal spread as revealed by conjugation experiments and de novo plasmid sequencing. These results show a complex pattern of AR persisting along the trophic gradient and an importance of anthropogenic influence on its dissemination in marine ecosystems, accentuating the imperative for comprehensive strategies congruent with the One Health paradigm to combat AR in the environment.