The ability of Staphylococcus aureus to form biofilms and persisters is a major cause of recalcitrant infections that are difficult to treat. We have examined time-dependent variation in persister population present in stationary-phase planktonic cells and biofilms of S. aureus when treated with bactericidal antibiotics having different cellular targets. Fourteen isolates identified as S. aureus were found to be resistant to three to nine classes of antibiotics tested according to the CLSI guidelines. Among the sensitive isolates, S48 was found to be the strongest biofilm producer, whereas J6 was the weakest. The four antibiotics, ciprofloxacin, daptomycin, tobramycin, and vancomycin, inhibited biofilm formation, whereas daptomycin was the strongest in disrupting 24-hr-old biofilm. Treatment of stationary-phase planktonic cells with 100 x minimum inhibitory concentration (MIC) of these antibiotics showed a typical biphasic pattern indicating the presence of persister cells. Twenty-four-hour-old biofilm of the two isolates tested at 100 x MIC of the antibiotics showed a similar biphasic pattern. Tolerance of biofilm cells was greater as compared with planktonic cells, which could be due to elevated number of persisters found in the biofilm as compared with planktonic cells.