ABSTRACT Vitek 2 (bioMérieux, Inc., Durham, NC) is a widely used commercial antimicrobial susceptibility testing system. We compared MIC results obtained by Vitek 2 to those obtained by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) reference method for 134 staphylococcal and 84 enterococcal clinical isolates. Nineteen agents were evaluated, including all those available on Vitek 2 for testing staphylococci and enterococci. The resistance phenotypes tested included methicillin-resistant Staphylococcus aureus (MRSA) ( n = 58), S. aureus with inducible clindamycin resistance (ICR) ( n = 30), trimethoprim-sulfamethoxazole-resistant MRSA ( n = 10), vancomycin-resistant Enterococcus ( n = 37), high-level gentamicin-resistant Enterococcus ( n = 15), linezolid-resistant Enterococcus ( n = 5), and daptomycin-nonsusceptible Enterococcus faecalis ( n = 6). For the staphylococci, there was 98.9% categorical agreement (CA). There was one very major error (VME) for gentamicin in a Staphylococcus hominis isolate, six VMEs for inducible clindamycin in S. aureus isolates, and two major errors (ME) for daptomycin in an S. aureus and a Staphylococcus epidermidis isolate. For enterococci, there was 97.3% CA. Two VMEs were observed for daptomycin in isolates of E. faecalis and 2 ME, 1 for high-level gentamicin resistance and 1 for nitrofurantoin, in E. faecium isolates. Overall, there was 98.3% CA and 99% essential agreement for the testing of staphylococci and enterococci by the Vitek 2. With the exception of detecting ICR in S. aureus , Vitek 2 performed reliably for antimicrobial susceptibility testing of staphylococci and enterococci.