Abstract Background: Antimicrobial resistance is growing substantially, which necessitates the search for novel therapeutic options. Terbinafne, an allylamine antifungal agent that exhibits a broad spectrum of activity and is used in the treat‑ ment of dermatophytosis, could be a possible option to disarm S. aureus virulence. Methods: Terbinafne inhibitory efect on staphyloxanthin was characterized by quantitative measurement of staphyloxanthin intermediates and molecular docking. The efect of terbinafne on S. aureus stress survival was charac‑ terized by viable counting. The anti‑bioflm activity of terbinafne on S. aureus was assessed by the crystal violet assay and microscopy. Changes in S. aureus membrane following treatment with terbinafne were determined using Fourier transform infrared (FTIR) analysis. The synergistic action of terbinafne in combination with conventional antibiotics was characterized using the checkerboard assay. qRT‑PCR was used to evaluate the impact of terbinafne on S. aureus gene expression. The infuence of terbinafne on S. aureus pathogenesis was investigated in mice infection model. Results: Terbinafne inhibits staphyloxanthin biosynthesis through targeting dehydrosqualene desaturase (CrtN). Docking analysis of terbinafne against the predicted active site of CrtN reveals a binding energy of − 9.579 kcal/mol exemplifed by the formation of H‑bonds, H‑arene bonds, and hydrophobic/hydrophilic interactions with the con‑ served amino acids of the receptor pocket. Terbinafne treated S. aureus was more susceptible to both oxidative and acid stress as well as human blood killing as compared to untreated cells. Targeting staphyloxanthin by terbinafne rendered S. aureus more sensitive to membrane acting antibiotics. Terbinafne interfered with S. aureus bioflm forma‑ tion through targeting cell autoaggregation, hydrophobicity, and exopolysaccharide production. Moreover, terbin‑ afne demonstrated a synergistic interaction against S. aureus when combined with conventional antibiotics. Impor‑ tantly, terbinafne attenuated S. aureus pathogenesis using mice infection model. qRT‑PCR revealed that terbinafne repressed expression of the transcriptional regulators sigB, sarA, and msaB, as well as icaA in S. aureus. Conclusions: Present fndings strongly suggest that terbinafne could be used safely and efciently as an anti‑viru‑ lent agent to combat S. aureus infections. Keywords: Staphylococcus aureus, Terbinafne, Staphyloxanthin, Bioflm, Virulence