Objective To evaluate the in vitro activity of daptomycin, vancomycin, teicoplanin, tigecycline, ceftobiprole and linezolid against 499 strains of blood-isolated gram-positive cocci. Methods Determination of the minimal inhibitory concentration (MICs) of daptomycin with microbrothdilution method and the MICs of other 9 antimicrnhial agents with agar dilution method against 499 strains of blood-isolated gram positive cocci was carried out. The data was analyzed with WHONET 5.4 software. Results The susceptibility rates of staphylococci to daptomycin, tigecycline, linezolid, ceftobiprole, vancomyein and teicoplanin were 100%. All staphylococcus strains were inhibited by daptomycin at a MIC of 1 mg/L. The MIC50 and MIC60 of daptomyein were both 0.5 mg/L against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus coagalase-negative (MRSCnN). Among Enterococcus spp, the highest MIC of daptomycin was 4 mg/L. The MIC50 and MIC90 of daptomycin were both 2 mg/L against E.faecalis, whereas they were 2 mg/L and 4 mg/L against E.faecium. One strains of linezolid-resistant E.faecalis(MIC:8 mg/L)was susceptible to daptomycin (MIC: 1 mg/L). Three strains of E.faecium carrying vanA gene with vancomycin MICs above 32 mg/L and teicoplanin MICs also 32 mg/L were susceptible to daptomycin, tigeeycline and linezolid. The MIC range of daptomycin against Streptococcus pneumoniae and Streptococcus viridans was 0.032-0.25 mg/L and 0.125-1.000 mg/L separately. Conclusions Daptomycin has excellent in vitro activity against common gram-positive pathogens isolated from blood. It may be a good choice for clinicians to treat drug-resistant gram-positive cocci.