The extensive use of broad-spectrum antibiotics in human medicine, farm animals, and the food industry has led to multidrug-resistant bacteria. The studies carried out within the scope of this Habilitation work aimed at resistance profiling and better understanding the mechanisms of resistance development and pathogenicity in the leading multiple drug resistance (MDR) bacterial pathogens known to cause nosocomial infections, such as ESKAPE pathogens, i.e. Acinetobacter baumannii, Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA), as well as bacteria causing neglected zoonoses such as Brucella. Combined in-vitro phenotyping with in-silico molecular analysis using Illumina MiSeq Next-Generation-Sequencing (NGS) technology and BioIT pipelines on a large number of clinical and non-clinical strains collected from Europe, Asia, and Africa were applied to achieve the goal of the One-Health approach. Antimicrobial resistance is a growing concern in veterinary medicine, the food chain, and the environment as in human medicine. However, data on animals, foods and environmental sources are still scarce. The emergence and dissemination of certain ESKAPE pathogens such as A. baumannii and K. pneumonia were reported since the late 20th century and increased over time in developed and developing countries. Whole-genome sequencing (WGS) is a powerful tool for rapidly identifying antimicrobial resistance (AMR) genes and can define resistance-associated determinants with much greater precision compared to conventional phenotypic tools. Continuous susceptibility testing, updating breakpoints, and assessing mutations that lead to resistance are needed for fastidious bacteria.