We used the broth microdilution method to determine the MICs of daptomycin and 13 comparator agents against 319 methicillin-susceptible Staphylococcus aureus isolates, 201 methicillin-resistant S. aureus (MRSA) isolates, and 183 coagulase-negative staphylococci (CoNS). Isolates were consecutively collected from bloodstream infections in 39 Spanish medical centers during a 3-month period (March through May 2008). Among MRSA, 1 isolate with intermediate susceptibility to vancomycin and 6 isolates resistant to linezolid were found. Nonsusceptibility to teicoplanin was detected in 3.9% of CoNS. Daptomycin was highly active against the staphylococcal blood isolates tested-all were inhibited at the daptomycin susceptibility breakpoint of < or = 1 microg/mL. Daptomycin retained its activity against the isolates that were resistant to teicoplanin or linezolid, or that had reduced susceptibility to vancomycin. These data suggest that daptomycin could be useful for the treatment of bloodstream infections caused by staphylococci.