OBJECTIVES: Increasing resistance to antimicrobials used for the treatment of Clostridioides difficile infections necessitates reproducible antimicrobial susceptibility testing. Current guidelines take a one-size-fits-all approach and/or offer limited guidance. We investigated how the choice of medium affects measured MIC values across two sites. METHODS: We determined MIC values for the antimicrobials fidaxomicin, metronidazole, and vancomycin for a representative collection of European C. difficile strains (n = 235) using agar dilution on three different media: Brucella Blood Agar (BBA), Fastidious Anaerobe Agar supplemented with horse blood (FAA-HB), and Wilkins-Chalgren (WC) agar. The study was conducted at two sites to compare reproducibility. Usability (ease of preparation of the media as well as read-out of the assay) was assessed through a survey. RESULTS: We found that all media result in highly consistent aggregated MIC data for all antibiotics, with MIC DISCUSSION: This study shows medium-dependent differences in C. difficile MICs for at least two antimicrobials across two sites. We suggest the use of FAA-HB to align with general European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations for susceptibility testing of anaerobic bacteria and deposited reference strains for standard susceptibility testing of C. difficile to increase interlaboratory reproducibility.