Staphylococcus aureus is a major cause of severe infections including infective endocarditis, but lineage-specific virulence determinants remain unclear. We analyzed 77 S. aureus bacteremic isolates from major lineages using phenotypic assays, infection models, and transcriptomics. Our results revealed significant heterogeneity in S. aureus pathogenicity. ST398 isolates exhibited heightened virulence, characterized by increased hemolysin production, whereas CC30 strains showed reduced growth, biofilm formation, and infectivity. Notably, the ST398 agrC mutant Sau7 exhibited unique phenotypic behavior, with high biofilm production and decreased virulence in Galleria mellonella larvae model. Infection studies in the rabbit experimental endocarditis model showed increased vegetation size and bacterial load in Sau7-infected animals, highlighting the role of agr system in S. aureus colonization and biofilm formation. Transcriptomic analysis identified key pathways, including quorum sensing systems and hemolysins, driving virulence in ST398 strains. These findings provide insights into the lineage-specific virulence mechanisms and the multifaceted nature of S. aureus pathogenicity.