Infections with multidrug-resistant (MDR) gram-positive bacteria are gradually increasing in the world. Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) are among the leading pathogens isolated from nosocomial infections. The increased use of vancomycin in the treatment has led to the development of isolates with reduced susceptibility and resistance. Daptomycin is a novel lipopeptide antibiotic which has a rapid bactericidal activity against infections caused by gram-positive bacteria including MRSA and VRE. The aim of this study was to evaluate the in vitro activity of daptomycin against VRE and MRSA strains isolated from clinical samples of hospitalized patients. A total of 229 isolates of which 118 were VRE and 111 were MRSA have been included in the study. All of the VRE strains were isolated from rectal swab samples and two of them were also resistant to linezolid with the MIC values of 8 µg/ml and 12 µg/ml. Bacterial identification was performed by conventional methods and susceptibility testing of daptomycin was performed by using the broth microdilution method as recommended by CLSI. The MIC values were interpreted according to CLSI susceptibility criteria for daptomycin. Daptomycin MIC50 and MIC90 values for VRE were found as 1 µg/ml and 2 µg/ml, respectively, with a MIC range of 0.125-2 µg/ml. Daptomycin MIC50 and MIC90 values for MRSA were determined as 0.12 µg/ml and 0.5 µg/ml, respectively, and the overall distribution of MIC values ranged between ≤ 0.032-1 µg/ml. All VRE including two linezolid-resistant strains and all MRSA strains (100%) were found susceptible to daptomycin. It was concluded that daptomycin is one of the few alternative agents for the treatment of infections caused by VRE and MRSA, nevertheless it was suggested that daptomycin MICs should better be monitored to prevent treatment failure due to the presence of non-susceptible strains and possible emergence of strains with decreased susceptibility during long-term therapy.