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The objective of this study was to determine minimum inhibitory concentrations (MIC) of frequently used antimicrobials for Escherichia coli and Trueperella pyogenes isolated from postpartum bovine uteri of cows with acute puerperal metritis (APM, n = 67), cows suspected to have APM (n = 37), and healthy cows (n = 37) and to evaluate possible differences in MIC according to clinical signs. Cows with APM had reddish-brown, fetid vaginal discharge and rectal temperature (RT) ≥39.5°C within 21 d in milk; cows suspected to have APM had either reddish-brown, fetid vaginal discharge or RT ≥39.5°C within 21 d in milk; and healthy cows had neither fetid discharge nor RT ≥39.5°C. Samples were collected from cows on commercial dairy herds (n = 7) using the cytobrush technique. A total of 37 T. pyogenes isolates and 85 E. coli isolates were tested. Ceftiofur, a third-generation cephalosporin that is often used to treat APM, was the focus of analysis. Trueperella pyogenes and E. coli were isolated more often from samples of cows with APM (46 and 90%, respectively) compared with samples from healthy cows (19 and 54%, respectively). Regarding cows suspected to have APM, T. pyogenes and E. coli were numerically more often isolated (30 and 70%, respectively) than in healthy cows (19 and 54%, respectively). Minimum inhibitory concentrations of ceftiofur were low. For T. pyogenes and E. coli, MIC50 (concentration that inhibited growth of 50% of isolates) were 0.25 and 0.5 µg/mL and MIC90 (concentration that inhibited growth of 90% of isolates) were 0.5 and 1 µg/mL, respectively. Although ceftiofur inhibited all T. pyogenes at the highest concentration tested (64 µg/mL), the growth of 5.9% of E. coli was not impaired. Recently, ampicillin has been suggested as an alternative treatment for APM. Although the T. pyogenes isolates exhibited low MIC in general (MIC50 ≤0.015 µg/mL and MIC90 = 0.06 µg/mL) and 81.1% of all T. pyogenes could be inhibited at the lowest ampicillin concentration tested, 11.8% of the E. coli isolates were not impaired at the highest concentration (64 µg/mL) tested in this study. The MIC50 and MIC90 of E. coli were 4 and ≥128 µg/mL, respectively. We detected no difference in the MIC distributions of ceftiofur or ampicillin among isolates from the 3 APM groups. In summary, E. coli with high MIC against ceftiofur as well as against ampicillin were found in this study.