Data regarding the evolution of Staphylococcus resistance in a whole country have a definite influence on the design of empirical treatment regimens. Nevertheless, incidence studies over long periods of time are expensive and very difficult to carry out. In order to ascertain the present situation of the antimicrobial resistance of Staphylococcus in Spain and the change of this resistance over time, we performed five point prevalence studies (1986 to 2002) in a large group of Spanish hospitals (from 68 institutions in 1986 to 143 in 2002) collecting all Staphylococcus strains isolated on a single selected day. All microorganisms were identified in the five studies at the same laboratory, and antimicrobial susceptibility testing was performed against 17 antimicrobial agents by the agar dilution method and a microdilution method. During this period, there was an overall increase in resistance to most antimicrobials among Staphylococcus aureus/coagulase-negative staphylococci, mainly to oxacillin (1.5%/32.5% in 1986 versus 31.2%/61.3% in 2002) (P < 0.001), erythromycin (7%/41.1% in 1986 versus 31.7%/63% in 2002) (P < 0.001), gentamicin (5.2%/25.4% in 1986 versus 16.9%/27.8% in 2002) (P < 0.001; P = 0.5), and ciprofloxacin (0.6%/1.1% in 1986 versus 33.9%/44.9% in 2002) (P < 0.001). All of the isolates were uniformly susceptible to glycopeptides, linezolid, and quinupristin/dalfopristin. Resistance of S. aureus to trimethoprim/sulfamethoxazole was very low (from 0.5% to 2.1%) (P = 0.152). Periodic performance of prevalence studies is a useful, inexpensive, and easy tool to know the nationwide situation of a microorganism and its resistance to antimicrobials; it also helps us assess the emergence and spread of antimicrobial resistance.