The spread of multidrug-resistant (MDR) bacterial infections has become a serious global threat. We introduce multi-layer coated gold nanoparticles (MLGNPs) delivering antisense oligonucleotides (ASOs) targeting the resistance gene of methicillin-resistant Staphylococcus aureus (MRSA), as a selective antimicrobial by restoring susceptibility. MLGNPs were prepared by multi-step surface immobilization of gold nanoparticles (GNPs) with polyethylenimine (PEI) and loaded with ASO targeting the mecA gene. The MLGNPs were shown to be efficiently internalized into various types of Gram-positive bacteria, including MRSA, Staphylococcus epidermidis, and Bacillus subtilis, which was superior to single-layer coated GNPs and free PEI polymer. The delivery of MLGNPs into MRSA resulted in up to 74% silencing of the mecA gene with high selectivity, in a dose-dependent manner. The treatment of MLGNPs to MRSA in the presence of oxacillin, a beta-lactam antibiotic, showed major suppression (~71%) of bacterial growth, due to the recovery of antibacterial sensitivity. Furthermore, the treatment of MLGNPs in a complex system showed preferential uptake into bacteria over mammalian cells, demonstrating the suitable characteristics of MLGNPs for selective delivery into bacteria. The current approach can be potentially applied for targeting various types of MDR bacterial infections by specific silencing of a resistance gene, as a combinatorial therapeutic used with conventional antibiotics.