Staphylococcus aureus is currently a significant multidrug-resistant bacterium, causing severe healthcare-associated and community-acquired infections worldwide. The current antibiotic regimen against this pathogen is becoming ineffective due to resistance, in addition, they disrupt the normal microbiota. It highlights the urgent need for a pathogen-specific drug with high antibacterial efficacy against S. aureus. α-Viniferin, a bioactive phytochemical compound, has been reported to have excellent anti-Staphylococcus efficacy as a topical agent. However, so far, there were no clinical trials that have been conducted to elucidate its efficacy. The present study aimed to investigate the antibacterial efficacy of α-viniferin against S. aureus in a ten-day clinical trial. Based on the results, α-viniferin showed 50% minimum inhibitory concentrations (MIC50 values) of 7.8 μg/ml in culture broth medium. α-Viniferin was administered in the nares three times a day for ten days using a sterile cotton swab stick. Nasal swab specimens were collected before (0 days) and after finishing the trial (10th day), and then analyzed. In the culture and RT-PCR-based analysis, S. ureus was reduced significantly: 0.01. In addition, 16S ribosomal RNA-based amplicon sequencing analysis showed that S. aureus reduced from 51.03% to 23.99% at the genus level. RNA-seq analysis was also done to gain insights into molecular mechanisms of α-viniferin against S. aureus, which revealed that some gene groups were reduced in 5-fold FC cutoff at two times MIC conditions. The study results demonstrate α-viniferin as a potential S. aureus-specific drug candidate.