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Abstract: Antibiotics are pivotal for treating Staphylococcus aureus infections, but overuse has led to multidrug-resistant (MDR) strains, necessitating alternative strategies. Targeting virulence factors, rather than bacterial survival, offers a promising approach to combat resistance. This study evaluated the in vitro effects of native, non-insecticidal Bacillus thuringiensis (Bt) parasporal crystal proteins (PCPs), in protoxin (PT) and trypsin-activated (AT) forms, on S. aureus virulence gene expression and biofilm formation. Fourteen Bt PCPs were isolated and prepared as PT and AT forms. Their impact was assessed using LacZ promoter fusion assays for hla (alpha-hemolysin), spa (protein A), and RNAIII (a key virulence regulator), supplemented by quantitative β-galactosidase assays. Biofilm formation was quantified and normalized to planktonic growth (Normalized Biofilm Index, NBI) to distinguish specific antibiofilm activity from growth inhibition. AT-PCPs from Bt strains M11, M78, M91, M154t1, and A11 significantly downregulated hla, spa, and RNAIII expression (log₂ fold changes up to –4.88, p < 0.001) and reduced biofilm formation (NBI reduction up to 57%, p < 0.001). AT proteins exhibited no hemolytic activity and had MIC values >100 μg/mL, confirming antivirulence effects at sub-inhibitory concentrations. PCR screening revealed the absence of cyt1 genes in four of five active isolates, supporting Cry-mediated targeting. These in vitro findings suggest that trypsin-activated PCPs can disrupt critical virulence pathways in S. aureus. Trypsin-activated Bt PCPs attenuate virulence and biofilm formation in S. aureus in vitro, highlighting their potential as leads for novel antivirulence strategies. These proteins show potential as candidates for standalone or adjuvant strategies alongside conventional antibiotics in the fight against MDR infections, pending further validation. Further research is needed to elucidate their mechanisms, assess their efficacy in vivo, and evaluate enzymatic activation by host-derived proteases. These in vitro results highlight the potential of Bt-derived PCPs as novel virulence-targeting leads, though further research is needed to identify the active component(s), elucidate mechanisms, and evaluate efficacy in vivo.