As a growing direction, nano-based therapy has become a successful paradigm used to address the phytogenic delivery-related problems in overcoming multivirulent vancomycin-resistant Staphylococcus aureus (VRSA) infection. Hence, our aim was to develop and assess a novel nanocarrier system (mesoporous silica nanoparticles, MPSNPs) for free berberine (Free-BR) as an antimicrobial alkaloid against strong biofilmproducing and multi-virulent VRSA strains using in vitro and in vivo mouse model. Results and discussion: Our outcomes demonstrated vancomycin resistance in 13.7% of Staphylococcus aureus (S. aureus) strains categorized as VRSA. Notably, strong biofilm formation was observed in 69.2% of VRSA strains that were all positive for icaA gene. All strong biofilm-producing VRSA strains harbored a minimum of two virulence genes comprising clfA and icaA with 44.4% of them possessing all five virulence genes (icaA, tst, clfA, hla, and pvl), and 88.9% being multi-virulent. The study findings affirmed excellent in vitro antimicrobial and antibiofilm properties of BR-loaded MPS-NPs. Realtime quantitative reverse transcription PCR (qRT-PCR) assay displayed the downregulating role of BR-loaded MPS-NPs on strong biofilm-producing and multivirulent VRSA strains virulence and agr genes in both in vitro and in vivo mice models. Additionally, BR- loaded MPS-NPs supplementation has a promising role in attenuating the upregulated expression of pro-inflammatory cytokines’ genes in VRSA-infected mice with attenuation in pro-apoptotic genes expression resulting in reduced VRSA-induced apoptosis. In essence, the current study recommends the future scope of using BR-loaded MPS-NPs as auspicious alternatives for antimicrobials with tremendous antimicrobial, antibiofilm, anti-quorum sensing (QS), and anti- virulence effectiveness against problematic strong biofilm-producing and multi- virulent VRSA-associated infections.