The prevalence of multidrug-resistant (MDR) bacteria has recently increased dramatically, seriously endangering human health. Herein, amoxicillin (Amoxi)-conjugated gold nanoparticles (AuNPs) were created as a novel drug delivery system to overcome MDR bacteria. MDR bacteria were isolated from a variety of infection sources. Phenotype, biotype, and 16S rRNA gene analyses were used for isolate identification. Additionally, Juniperus excelsa was used for the production of AuNPs. The conjugation of AuNPs with Amoxi using sodium tri-polyphosphate (TPP) as a linker to produce Amoxi-TPP-AuNPs was studied. The AuNP and Amoxi-TPP-AuNP diameters ranged from 15.99 to 24.71 nm, with spherical and hexagonal shapes. A total of 83% of amoxicillin was released from Amoxi-TPP-AuNPs after 12 h, and after 3 days, 90% of the medication was released. The Amoxi-TPP-AuNPs exhibited superior antibacterial effectiveness against MRSA and MDR E. coli strains. Amoxi-TPP-AuNPs had MICs of 3.6–8 µg mL⁻¹ against the tested bacteria. This is 37.5–83 fold higher compared to values reported in the literature. Amoxi-TPP-AuNPs exhibit a remarkable ability against MRSA and E. coli strains. These results demonstrate the applicability of Amoxi-TPP-AuNPs as a drug delivery system to improve therapeutic action.