Staphylococcus aureus can cause a wide range of severe infections owing to its multiple virulence factors in addition to its resistance to multiple antimicrobials; therefore, novel antimicrobials are needed. Herein, we used Gardenia thailandica leaf extract (GTLE), for the first time for the biogenic synthesis of silver nanoparticles (AgNPs). The active constituents of GTLE were identified by HPLC, including chlorogenic acid (1441.03 μg/g) from phenolic acids, and quercetin-3-rutinoside (2477.37 μg/g) and apigenin-7-glucoside (605.60 μg/g) from flavonoids. In addition, the antioxidant activity of GTLE was evaluated. The synthesized AgNPs were characterized using ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, transmission and scanning electron microscopy (SEM), zeta potential, dynamic light scattering, and X-ray diffraction. The formed AgNPs had a spherical shape with a particle size range of 11.02–17.92 nm. The antimicrobial activity of AgNPs was investigated in vitro and in vivo against S. aureus clinical isolates. The minimum inhibitory concentration (MIC) of AgNPs ranged from 4 to 64 µg/mL. AgNPs significantly decreased the membrane integrity of 45.8% of the isolates and reduced the membrane potential by flow cytometry. AgNPs resulted in morphological changes observed by SEM. Furthermore, qRT-PCR was utilized to examine the effect of AgNPs on the gene expression of the efflux pump genes norA, norB, and norC. The in vivo examination was performed on wounds infected with S. aureus bacteria in rats. AgNPs resulted in epidermis regeneration and reduction in the infiltration of inflammatory cells. Thus, GTLE could be a vital source for the production of AgNPs, which exhibited promising in vivo and in vitro antibacterial activity against S. aureus bacteria.