Objectives Bacillus anthracis clinical breakpoints, representing a systematic approach to guide clinicians in selecting the most appropriate antimicrobial treatments, are not part of the guidance from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). This is because defined distributions of MIC values and of epidemiological cut-off values (ECOFFs) have been lacking. In this study, a Europe-wide network of laboratories in collaboration with EUCAST, aimed at establishing standardized antimicrobial susceptibility testing (AST) methods, wild-type (WT) MIC distributions and ECOFFs for ten therapeutically relevant antimicrobials. Methods About 335 B. anthracis isolates were tested by broth microdilution (BMD) and disc diffusion (DD) methodologies. Minimum inhibitory concentrations (MIC) and inhibition zone diameters were curated according to EUCAST SOP 10.2 and the results were submitted to EUCAST for ECOFFs and clinical breakpoint determination. Results BMD and DD data distributions revealed putative WT distributions for the tested agents. For each antimicrobial agent, ECOFFs were defined. Three highly resistant strains with MIC values of 32 mg/L benzylpenicillin were found. MIC values slightly above the defined ECOFFs were observed in a few isolates, indicating the presence of resistance mechanisms to doxycycline, tetracycline and amoxicillin. Conclusions B. anthracis AST results were used by EUCAST to determine ECOFFs for ten antimicrobial agents. The MIC distributions were used in the process of determining clinical breakpoints. The ECOFFs can be used for the sensitive detection of isolates with resistance mechanisms, and for monitoring resistance development. Genetic changes causing phenotypic shifts in isolates displaying slightly elevated MICs remain to be investigated.