Acinetobacter baumannii is recognised as a priority 1 critically important pathogen by the World Health Organisation. The cow rumen has previously yielded antimicrobial peptides namely Lynronne-1, -2 and -3 with high efficacy against bacterial pathogens, such as Staphylococcus aureus and Pseudomonas aeruginosa. In this study we assessed the structure by circular dichroism and efficacy of Lynronne-1, -2 and -3 against clinical strains of A. baumannii. Lynronne-1, -2 and -3 demonstrated alpha-helical secondary structures and had antimicrobial activity towards all tested strains of A. baumannii (Minimum Inhibitory Concentrations 2-128 μg/ml). Lynronne-2 and -3 also demonstrated additive effects with amoxicillin and erythromycin, and synergy with gentamicin. The AMPs demonstrated little toxicity towards mammalian cell lines or Galleria mellonella. Antibiofilm activity was observed with all three AMPs. Lynronne-1 and -3 demonstrated higher membrane-destabilising action against A. baumannii in comparison with Lynronne-2 in a fluorescence-based assay. This was corroborated by transcriptomic analysis which highlighted several gene expression changes related to cell wall synthesis following addition of the AMPs. For the first time we demonstrate the therapeutic activity of Lynronne AMPs against A. baumannii.