Klebsiella pneumoniae causes common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance (MDR) and mortality. In this study, we investigated the ability of the antisense peptide nucleic acids (PNA) conjugated to the (KFF)3K cell-penetrating peptide (CPP) to target the gyrA KPC-producing K. pneumoniae and inhibit bacterial growth in vitro. The inhibitory effect on gyrA gene was evaluated by measuring 16s gene amplification in KPC-producing K. pneumoniae treated with the antisense PNA conjugate. The hemolytic property of the antisense PNA conjugate was accessed toward mice red blood cells. Finally, molecular modeling and dynamics simulations analyses in aqueous solutions were performed to predict the PNA conformation alone in contact with DNA (gyrA gene sequence). PNA was capable of inhibiting bacterial growth at 50 μM, also reducing 16S gene amplification in 96.7%. Besides, PNA presented low hemolytic activity (21.1% hemolysis) at this same concentration. Bioinformatics analysis demonstrated that the structure of the PNA is stable in water without major changes in its secondary structure. The ability of PNA and its conjugated CPP ((KFF)3K) to inhibit bacterial growth demonstrates the potential of this new class of antibacterial agents, encouraging further in vivo studies to confirm its therapeutic efficacy.