Abstract <p>This study investigated the persistence of small colony variants (SCVs) within methicillin resistant <i>Staphylococcus aureus</i> (MRSA) biofilms. SCVs are dormant bacterial subpopulations that emerge under stress and contribute to chronic infections. Ten MRSA isolates were examined, all of which formed biofilms containing a mixed population of metabolically active wild-type (WT) cells and small colony variants (SCVs). The SCVs, which primarily localized at the biofilm base, are characterized by three key features: (1) decreased metabolic activity, (2)&#xa0;down-regulated expression of critical virulence and communication genes, and (3) enhanced adhesiveness. These features suggest SCVs enter a dormant state that enables extended persistence within biofilms, potentially surviving harsh conditions that would eliminate WT cells. This dormancy comes with trade-offs: SCVs depend on external factors to reactivate their metabolism. Recovery assays showed decreasing recovery rates for both WT and SCVs as biofilms aged, with WT maintaining higher viability throughout the 45-day incubation period. Atomic force microscopy (AFM) revealed SCVs possess stiffer cell walls (51.6 pN/nm) compared to WT cells (23.6 pN/nm), with stiffness increasing with biofilm age. Both phenotypes showed time-dependent increases in surface adhesion, but SCVs demonstrated a more pronounced enhancement (42% increases vs. 30% for WT by day 45), suggesting stronger integration with the maturing biofilm matrix. Notably, these phenotypes may remain surface-associated even after cell death.</p>