The purpose of this study is to establish a method for assessing the anaerobic environment for EUCAST disk diffusion antimicrobial susceptibility testing (AST) of anaerobic bacteria on fastidious anaerobe agar with 5% mechanically defibrinated horse blood (FAA-HB). The method utilizes the association between a decrease in the metronidazole disk zone diameter and increasing oxygen levels with an aerotolerantClostridium perfringensstrain DSM 25589 (CCUG 75076 and NCTC 14679). TheC. perfringensstrain was tested on FAA-HB with a McFarland 1 inoculum and a metronidazole 5 μg disk. FAA-HB was incubated for 16–20 h at 35–37°C. The association between oxygen levels (0, 0.16, 1, 2, and 4% oxygen) and the metronidazole zone diameter was determined. Reproducibility at 0% oxygen was investigated as part of a European multi-centre study of disk diffusion of anaerobic bacteria. The median zone diameters (n=12) at each oxygen level were 29 mm (0%), 21 mm (0.16%), 16 mm (1%), 15 mm (2%), and 15 mm (4%). The metronidazole zone diameters at 0% oxygen from the multi-centre reproducibility-study had a median of 29 mm and a 95%-percentile range of 25–33 mm (n=236). Only one reading was below 25 mm. Based on our results, a zone diameter of ≥25 mm using a metronidazole 5 μg disk and theC. perfringensstrain, tested with EUCAST recommendations, can be used to indicate that the anaerobic environment is of sufficient quality for culture and disk diffusion AST. EUCAST has included the method as part of the quality control for AST of anaerobic bacteria.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10096-023-04622-9.