Abstract Background Few studies report the minimum inhibitory concentrations for antimicrobials against equine Corynebacterium pseudotuberculosis isolates. Hypothesis/Objectives To evaluate trends in the in vitro activities of 20 antimicrobials against equine Corynebacterium pseudotuberculosis isolates from 1996 to 2012 and to determine if a relationship exists between the minimum inhibitory concentration (MIC) and location of the abscess. Animals Corynebacterium pseudotuberculosis isolates from 196 horses with naturally occurring disease. Methods Retrospective and cross-sectional design. Medical records were reviewed to obtain clinical and MIC data. Minimum inhibitory concentrations were determined by the microdilution technique. The MIC results over 3 periods were compared (1996–2001, 2002–2006, 2007–2012). Results The MIC90 values for clinically relevant antimicrobials were as follows: chloramphenicol ≤4 μg/mL, enrofloxacin ≤0.25 μg/mL, gentamicin ≤1 μg/mL, penicillin =0.25 μg/mL, rifampin ≤1 μg/mL, tetracycline ≤2 μg/mL, trimethoprim-sulfamethoxazole (TMS) ≤0.5 μg/mL, ceftiofur =2 μg/mL, and doxycycline ≤2 μg/mL. There were no significant changes in MIC results over the study period. There was no relationship between MIC patterns and abscess location. Conclusions and Clinical Importance The MIC50 and MIC90 values of antimicrobials evaluated in this study for equine isolates of C. pseudotuberculosis did not vary over time. Abscess location was not associated with different MIC patterns in cultured isolates. Several commonly used antimicrobials are active in vitro against C. pseudotuberculosis in vitro.