Acinetobacter spp. are a group of Gram-negative bacteria found in a variety of different environments such as hospitals, food, soil and water sources. In fact, investigation of Acinetobacter spp. has been declared a One Health crisis. highlighting that not just clinical, but also animal and environmental samples are important to our understanding and resolution of these infections. Many strains of the most notorious member, Acinetobacter baumannii are multi-drug resistant (MDR), and virulent, causing infections in humans including soft tissue and wound infections, ventilator-associated pneumonia and even bacteremia. Various antibiotic resistance genes (ARGs) are common in Acinetobacter spp. In Chapter 3, the diversity of Acinetobacter spp. is highlighted with the discovery of novel sequence types and therefore novel clonal complexes from non-clinical sources. We found that the most common ARGs are efflux pumps and the presence of ARGs did not necessarily determine antibiotic susceptibility. There was also no correlation between virulence and geographic origin or isolation source. In Chapter 4, we explore clinical MICs of colistin resistance observed in clinical isolates of Acinetobacter courvalinii and Acinetobacter pittii, and in non-clinical isolates of Acinetobacter seifertii. Again, with Chapter 3 as a basis, Chapter 5 describes the discovery of an ADC-5 cephalosporinase encoding gene from a tank milk isolate of A. baumannii, the first to be observed in a non-clinical isolate. Efflux pumps were the most commonly detected ARG and this was studied in more detail in Chapter 6. Determinants of substrate selectivity using molecular genetic techniques. Finally, because of the classification of A. baumannii as a One Health challenge. understanding the impact of a wide-spread environmental non-antibiotic compound, SA, and its effects on antibiotic susceptibility and virulence are explored in Chapter 7. Studying the impact of SA on ARGs and virulence genes (VGs) using a proteomic approach revealed changes in membrane specific structures such as efflux pumps and porins thereby affecting antibiotic susceptibility. By taking a One Health approach to studying ARGs and VGs in non-clinical and clinical Acinetobacter spp, this study greatly contributes to the understanding of resistance and virulence mechanisms and non-antibiotic triggers thereby contributing to prevention and treatment of such infections.