Staphylococcus aureus is a commensal bacterium that asymptomatically colonizes humans. However, S. aureus could be an extremely versatile pathogen in humans causing life-threatening infections. S. aureus possesses a wide range of virulence factors that enable it to thrive within the host. The current study intended to illustrate the influence of both celastrol and terbinafine on S. aureus staphyloxanthin biosynthesis, virulence determinants expression as well as host pathogenesis. A total of 100 S. aureus isolates were recovered from 190 specimens during the period from June 2019 to December 2019 from patients admitted to the El-Ahrar Educational Hospital and Zagazig university Hospital, Zagazig, Sharkia, Egypt. All S. aureus clinical isolates were subjected to carotenoid pigment extraction and quantification. Factors affecting staphyloxanthin production in S. aureus were assessed and staphyloxanthin production was showed to be maximized in trypticase soya broth medium. The long incubation time reduced pigment production. Additionally, increased aeration also enhanced staphyloxanthin biosynthesis. The inhibitory activity of celastrol and terbinafine on S. aureus staphyloxanthin biosynthesis, production of virulence determinants as well as host pathogenesis was fully characterized. Celastrol and terbinafine efficiently reduced staphyloxanthin biosynthesis in S. aureus both the standard strain and clinical isolates recovered from different sources such as wound, burn, endotracheal aspirate and urine. Quantitative measurement of staphyloxanthin intermediates upon celastrol treatment showed a significant reduction in staphyloxanthin as well as its intermediates, including 4,4`-diapophytoene, 4,4`-diaponeurosporene and 4,4`-diaponeurosporenic acid as compared to untreated cells. High performance liquid chromatography (HPLC) showed an extra peak in carotenoid pigments extracted from celastrol-treated S. aureus compared tountreated bacteria, which was further identified as farnesyl diphosphate by mass spectrometry. Importantly, the molecular docking study further validated that celastrol interacts with CrtM active sites with a binding energy of - 9.15 kcal/mol. On the other hand, treatment of S. aureus with terbinafine results in a significant accumulation of the CrtN substrate; 4,4`-diapophytoene, with significant reduction of the subsequent staphyloxanthin biosynthesis intermediates (4,4`-diaponeurosporene and 4,4`-diaponeurosporenic acid) as compared to untreated bacteria. Mass spectrometry showed accumulation of 4,4`-diapophytoene upon terbinafine treatment. Moreover, the molecular docking analysis validated terbinafine interaction with CrtN with a characteristic binding mode and affinity, exhibiting a binding energy of -9.579 kcal/mol. Importantly, celastrol treatment significantly decreased S. aureus survival to oxidative stress, acidic stress and whole blood killing when compared to untreated cells. Similarly, terbinafine-treated S. aureus were more susceptible to hydrogen peroxide, acid stress and whole blood killing when compared to untreated. Inhibition of staphyloxanthin upon celastrol and terbinafine altered S. aureus cell membrane rigidity as revealed by Fourier infrared spectroscopy (FTIR) analysis. This altered membrane rigidity rendered S. aureus cells more susceptible to membrane targeting antibiotics such as polymxin B. In addition to the staphyloxanthin biosynthesis inhibitory potential, celastrol and terbinafine exhibit a significant anti-biofilm activity against S. aureus as indicated by crystal violet assay and microscopy. Both celastrol and terbinafine significantly reduced biofilm mass on polystyrene and polypropylene surfaces in a dose dependent manner. Furthermore, celastrol and terbinafine treatment rendered S. aureus cells dispersed with minimal aggregates, deficient exopolysaccharide and altered cell hydrophobicity.Interestingly, celastrol and terbinafine markedly synergized the action of conventional antibiotics such as ampicillin, cefotaxime, ciprofloxacin, azithromycin, and gentamicin against S. aureus with fractional inhibitory concentration index (FICI) ranging from 0.12 to 0.31 and 0.14 to 0.31, respectively. The expression levels of genes responsible for staphyloxanthin biosynthesis (crtM and crtN), biofilm formation (icaA, icaR, sarA and agrA), oxidative stress response (katA, sodA and sodM), transcription regulation (msaB, sigB) and stress adaptation (yjbH), were determined in both celastrol and terbinafine treated and untreated S. aureus cells using RT-qPCR. The results demonstrate that celastrol could alter the expression of staphyloxanthin biosynthesis genes (crtM and crtN) as well as biofilm and virulence genes. However, terbinafine did not affect the expression of staphyloxanthin biosynthesis genes while repressed the expression of the virulence regulators sigB, sarA, msaB and icaA in S. aureus. Celastrol and terbinafine effect on S. aureus virulence and pathogenesis was assessed using in vivo mice infection model. Mice organs infected with pigmented bacteria were congested and copious with S. aureus cells than those isolated from mice injected with celastrol, terbinafine-treated bacteria or non-pigmented S. aureus. Furthermore, histopathological examination of organ tissues isolated from mice infected with pigmented S. aureus revealed abnormal tissue pathology with irreversible tissue necrosis and fibrosis. In contrast, organ tissues isolated from mice infected with celastrol, terbinafine-treated and non-pigmented S. aureus showed immature lesions with mild inflammatory signs and minimal tissue pathological changes. In conclusion, current study highlights the influence of both celastrol and terbinafine on S. aureus staphyloxanthin biosynthesis, virulence determinants production as well as host pathogenesis. S. aureus staphyloxanthin biosynthesis and biofilm formation were significantly reduced in the presence of eithercelastrol or terbinafine. Furthermore, both celastrol and terbinafine increased S. aureus susceptibility to environmental stresses and conventional antibiotics. S. aureus virulence and host pathogenesis was significantly disrupted upon treatment with either celastrol or terbinafine. Present data further support the efficiency of celastrol and terbinafine in treatment of S. aureus infections. The current study suggests the involvement of celastrol and terbinafine in treatment protocols in order to control and manage infections caused by against S. aureus