Acinetobacter baumannii is a multi-drug resistant Gram-negative bacterium of global concern. It commonly causes nosocomial infections such as ventilator-assisted pneumonia, bacteraemia, urinary tract infections, etc. The genome of AB5075 consists of three plasmids (83 kb, 8.7 kb and 2 kb) and a ~4 Mbp chromosome. The largest of the three plasmids “p1AB5075” harbors several antibiotic resistance genes (ARGs) e.g., aadB and strA and numerous unidentified genes. Unfortunately, this plasmid has only been studied in terms of antimicrobial resistance specifically, investigating resistance island 2 (RI-2) to date. In this research we demonstrate that upon the loss of p1AB5075 there is a reduction of motility, adhesion capability to polystyrene plastic and an increase in biofilm formation. We hypothesized that the second copy of H-NS located on p1AB5075 is responsible for the observed change in biofilm formation. Upon reintroduction of the plasmid-encoded H-NS into the Δp1AB5075 background, biofilm formation levels are restored to wildtype levels. The addition of plasmid-encoded H-NS to Δp1AB5075 and wildtype strains reveals a significant growth defect in L-broth and minimal media. RNA-seq revealed evidence of severe dysregulation of cellular processes that may explain this reduction in bacterial fitness. Overall, the p1AB5075-borne copy of H-NS influences biofilm production in A. baumannii. This is important as dissemination of this plasmid into the environment may not alter just the antibiotic resistance profile but also, the regulation of chromosomally-encoded biofilm associated genes already present in the recipient strain due to the presence of the global transcriptional regulator H-NS.