A previous transcriptome study revealed an increase in antisense transcription and gene expression changes in the absence of the transcription termination factor Rho in Staphylococcus aureus. This study assessed the physiological importance of Rho-dependent transcription termination by comparing the S. aureus HG001 strain and its isogenic rho-deletion mutant. Proteome analysis revealed an increase in the levels of secreted virulence factors that are controlled by the SaeRS two-component system in the absence of Rho in vitro. In addition, inhibition of Rho by the antibiotic bicyclomycin led to increased levels of SaeRS-dependent virulence factors, similar to those observed in the rho-deletion mutant. Finally, a rho-deletion strain exhibited increased virulence in mice compared to the wild type. These findings identify a link between Rho-dependent transcription termination and virulence regulation in S. aureus and suggest that antibiotic treatment can modulate the expression of virulence factors.