For optimum activity of daptomycin (DAP) in vitro, medium supplemented with calcium ions at physiological concentration (i.e., 50 mg/l) is required for determination of DAP minimum inhibitory concentration (MIC) in the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) method. However, our literature review found that Mueller–Hinton agar (MHA) brands used for the DAP Etest had different calcium ion (Ca2+) concentrations among the reports. For 98 clinical methicillin-resistant Staphylococcus aureus isolates previously unexposed to DAP, MICs were assessed by use of the Etest with MHA plates with different media (MHA-A, MHA-B, and MHA-C) and compared with those from the CLSI reference BMD method. The instructions for the Etest recommend MHA with Ca2+ of 25–40 mg/l for DAP MIC testing; Ca2+ concentrations for each type of MHA were 20.4 mg/l in MHA-A, 45.2 mg/l in MHA-B, and 52.0 mg/l in MHA-C. When the MIC50/MIC90 of the clinical isolates were studied, the Etest MICs for MHA-A were 1-fold dilution higher than for the BMD value. In contrast, for MHA-B they were 1-fold dilution lower, and for MHA-C MIC50 was 1.5-fold dilution lower and MIC90 was 2-fold dilution lower. MICs measured in MHA with higher Ca2+ tended to be lower. Comparison of MICs between BMD and the Etest for each MHA showed they were significantly different (p < 0.0001). The correlation coefficient was 0.6258 (p < 0.0001) for MHA-A, 0.4224 (p < 0.0001) for MHA-B, and 0.2504 (p = 0.0129) for MHA-C. Our results suggest there are differences in DAP MICs between MIC testing methods and differences between Ca2+ concentrations in MHA. For more objective and accurate measurement of DAP MICs, there should be discussion about standardization of Ca2+ in MHA.