Staphylococcus aureus can cause persistent infections and is known to develop persister cells in vitro. However, the in vivo significance of in vitro persisters in general is largely unclear. Here, we evaluated S. aureus stationary phase cultures and biofilm bacteria enriched in persister bacteria in comparison with actively growing log phase bacteria in terms of their ability to cause disease in a mouse skin infection model. We found that mice infected with the stationary phase and biofilm bacteria, which were enriched with persisters, produced more pronounced skin lesions that took longer to heal, and had more severe skin pathology and higher bacterial load than mice infected with log phase bacteria. Using our persistent infection mouse model, we showed that the clinically recommended treatment for recurrent S. aureus skin infection, doxycycline + rifampin, was not effective in eradicating the bacteria in mice. Analogous findings were observed in a Caenorhabditis elegans model, where stationary phase S. aureus caused greater virulence or mortality than log phase bacteria as early as two days post-infection. Our findings associate in vitro persisters and biofilm bacteria with more persistent and more severe infections and emphasize the importance of quality or metabolic status of the inoculum bacteria (persister bacteria versus growing bacteria) not just the number of bacteria in causing disease. The persistent infection mouse model we developed with persister inocula should have implications for understanding the process of disease establishment and pathogenesis, for developing persistent infection animal models, and for developing more effective treatments for chronic persistent infections in general.