MIC testing was performed simultaneously by Etest and broth microdilution (BMD) on 587 Staphylococcus aureus isolates submitted by local laboratories to a reference laboratory for confirmatory testing (May 2005 to July 2008). Testing bias was assessed for Etest to BMD MIC ratios. Categoric and essential agreement, very major (BMD nonsusceptible, Etest susceptible), and major (BMD susceptible, Etest nonsusceptible) errors were evaluated. Agar and broth calcium concentrations were consistent with current Clinical and Laboratory Standards Institute and manufacturer recommendations. There was a consistent bias for higher Etest MIC values compared with BMD. Ratios ranged from 0.25 to 4 (average 1.3), with substantial variability noted among the 8 different Etest lots tested. Overall, 6% of all ratios were >2.0. Categoric agreement and essential agreement among the 8 Etest lots ranged from 73% to 96% and 74% to 100%, respectively; very major errors ranged from 3% to 9%, and major errors ranged from 6% to 35%. However, most of the discrepancies were limited to 3 Etest lots.