Background: Infections affecting neonates caused by Staphylococcus aureus are widespread in healthcare facilities; hence, novel strategies are needed to fight this pathogen. Targeting virulence factors necessary to induce host damage and disease is a new tactic for antimicrobial therapy with several benefits which could lead to decreased resistance. The effect of FDA-approved drugs (sodium bicarbonate, dexamethasone, and ascorbic acid) with sub-inhibitory concentration (1/8 MIC) was evaluated for their capability to hinder virulence factors production in Staphylococcus aureus phenotypically. Furthermore, their effects were estimated genotypically using qRT‑PCR.Results: Sub-inhibitory concentrations (1/8 MIC) of sodium bicarbonate, dexamethasone, and ascorbic acid reduced the production of Staphylococcus aureus virulence factors, including biofilm formation, staphyloxanthin, proteases, and hemolysin production, as well as resistance to oxidative stress. At the molecular level, qRT-PCR was used to assess the relative expression levels of CrtM, SigB, SarA, AgrA, hla, fnbA, and icaA genes regulating virulence factors production and showed a significant reduction in the relative expression levels of all the tested genes. Conclusions: In summary, the current findings reveal that sodium bicarbonate, dexamethasone, and ascorbic acid have strong anti-virulence effects against multi-drug resistant Staphylococcus aureus. Thus, suggesting the use of sodium bicarbonate, dexamethasone, and ascorbic acid for fighting Staphylococcus aureus, and also suggesting that they might be used to treat infections caused by multi-drug resistant Staphylococcus aureus in place of or in addition to conventional antimicrobials.