The increasing global health threat posed by antibiotic resistance is diminishing the effectiveness of treatments for bacterial infections, particularly with the emergence of multidrug-resistant pathogens. These "superbugs," especially prevalent in hospital environments, pose significant challenges to healthcare systems. Acinetobacter baumannii, recognised by the World Health Organization (WHO) as a high-priority pathogen, exemplifies the critical need for new strategies to combat these resistant bacteria. Multidrug efflux pumps play a central role in bacterial drug resistance, making them key targets in the effort to restore the efficacy of antimicrobial treatments. In a recent TraDIS biocide screening, we identified a novel transmembrane protein, Neo2, as a significant contributor to silver nitrate biocide tolerance in A. baumannii. To further investigate Neo2’s role in antimicrobial resistance, we conducted whole-cell transport assays and employed the Biolog Phenotypic MicroArray (PM) assay for substrate profiling. We also examined the growth effects of cells expressing Neo2 when exposed to various antimicrobial compounds. Our study revealed that Neo2 markedly enhances tolerance to ethanol and the last-resort antibiotics such as colistin (polymyxin E) and polymyxin B. Given that these compounds primarily target the cell envelope, we hypothesise that Neo2 is crucial in maintaining cell membrane integrity. Ongoing experiments, including lipidomics, cell permeability assays, flow cytometry, and oxidative stress tolerance studies, are currently underway to evaluate Neo2’s impact on the cell envelope integrity of A. baumannii. Additionally, homologs of Neo2 are found in other top-priority pathogens identified by the WHO. The discovery of Neo2 as a key factor in tolerance against the last resort antibiotics is very significant and has the potential to act as a novel therapeutic determinant in the fight against multidrug-resistant bacterial pathogens.