No report exists on the role of Mastoparan B (MP-B) as an RND efflux pump inhibitor in multi-drug resistant (MDR) Acinetobacter baumannii. Here, we performed a series of in-silico experiments to predict the inhibition of the AdeB efflux pump by MP-B as a drug target agent. For this reason, an MDR strain of A. baumannii was subjected to minimum inhibitory concentration (MIC) against 12 antibiotics as well as MP-B. Expression of the a deB gene in the absence and presence of sub-MIC of MP-B was studied by qRT-PCR. It was found that MP-B had potent antimicrobial activity (MIC=1 µg/ml) associated with a 20-fold decrease in its expression at sub-MIC of MP-B. The stereochemical analysis using several automated servers confirmed that the AdeB is an inner membrane of the RND tripartite complex system with helix-turn-helix conformation and a pore rich in Phe, Ala, and Lys residue. The best model that showed high accuracy (Z=1.2, C=1.41, TM=0.99, and RMSD=4.4) was selected for docking purposes using the Site Map tool, and the correct protein-peptide complexes were simulated in the BioLiP platform. The molecular docking via AutoDock/Vina suggested that MP-B form H-bound with amino acid residues of the AdeB helix-5 and caused a shift in the dihedral angle by distances of 9.0 Å, 9.3 Å, and 9.6 Å, respectively. This shift was detected by the AlphaFold 2 tool and influenced the overall druggability of the protein. From the results, we concluded that, MP-B can be a good candidate for inhibition of bacterial efflux pump.