Several multidrug-resistant organisms have emerged, which increases the threat to public health around the world and a limited number of therapeutics were available to counteract these issues. Thus, researchers are trying to find out novel antimicrobials to overcome multidrug-resistant issues. The present study aimed to isolate antibacterial principles against the clinical isolates of multidrug-resistant (MDR)Staphylococcus aureusfrom the ethyl acetate extract ofGycosmis pentaphylla. The isolation and structural characterization of bioactive compounds were carried out using various chromatographic techniques (TLC, column, HPLC, and LC-MS) and spectral studies such as FT-IR, CHNS analysis,H-1-NMR, and(13)C-NMR. The antimicrobial potential of isolated compounds was assessed according to the standard methods. The isolated compounds were identified as arborine and skimmianine, which exhibited a significant antibacterial effect with the lowest MIC and MBC values against MDRS. aureusandin vitrokinetic and protein leakage assays supported the antimicrobial activity. Significant morphological changes such as uneven cell surfaces and morphology, cell shrinkage, and cell membrane damages were observed in the MDRS. aureusupon the treatment of arborine and skimmianine. The present investigation concludes that the isolated arborine and skimmianine compounds fromG. pentaphyllaharbor a strong antibacterial activity against MDRS. aureusand may be used as alternative natural drugs in the treatment of MDRS. aureus.