Antimicrobial resistance (AMR) is one of the greatest threats to human health and requires a comprehensive One Health approach for thorough investigation and control. However, surveillance of AMR in bacteria is limited to the clinical setting both in Croatia and worldwide, while little attention is paid to the environment. Despite the important role of groundwater as a drinking water supply, its contribution to the global AMR crisis is still largely unexplored. To fill this gap, the ARES project will adopt a holistic, interdisciplinary approach to investigate the resistome, microbiome and mobilome of four groundwater sources, including three karst sources used for water supply in Croatia. We will apply culture-independent methods such as high-throughput qPCR arrays and standard qPCR in combination with 16S rRNA amplicon sequencing to investigate the resistome and microbiome of groundwater over several seasons. This will allow us to assess temporal trends in the diversity and abundance of the microbiome and resistome in the studied groundwater sources. In addition, we will use culture-dependent approaches to assess the AMR profile of clinically important pathogens (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterococcus spp.) isolated from groundwater and investigate the molecular mechanisms of their resistance and virulence potential using whole-genome sequencing. Finally, the direct capture of antibiotic-resistant plasmids from groundwater bacteria to E. coli and their subsequent complete sequencing will be crucial to draw conclusions on the potential exchange of AMR between environmental reservoirs and the community. We believe that this knowledge will provide important insights for management strategies aimed at mitigating the emergence and spread of AMR and important human pathogens via drinking water reservoirs.