The Penicillin Binding Proteins (PBPs) are important biological target for new antibacterial drugs development. This study focused on molecular interaction between cefoxitin and the Escherichia coli PBP5 by molecular dynamics (MD) by using hybrid quantum mechanics/molecular mechanics (QM/MM) simulations approach, searching to develop a computational simulations prototype method on antimicrobial susceptibility of gram-negative bacteria against antibiotics. E. coli ATCC 8739 strain susceptibility for the drugs used in the antimicrobial susceptibility testing and selection of bioactive molecules against resistant strain. The protonation revealed a deprotonate state for His146, His151, His216 and His320 residues. The complex was stabilized after 0.6 ns of MD simulation. The global interaction means for inhibition zone diameters of E. coli ATCC8739 strain and cefoxitin was 24.33 mm no showing significant difference between computational and experimental methods. Our computational simulation method can reliably be performed as a molecular modeling prototype for gram-negative antimicrobial susceptibility testing bacteria. This article is protected by copyright. All rights reserved.