Objectives Disc diffusion is a cost-efficient, low-complexity, reliable method for detection of blaZ -mediated benzylpenicillin resistance in Staphylococcus aureus if the zone edge is inspected. EUCAST breakpoints cannot fully separate β-lactamase-positive from β-lactamase-negative strains, and EUCAST recommends the zone edge test. Literature on nitrocefin-based testing and the zone edge test is scarce with wide variations in reported assay performance. Methods This study compared two different nitrocefin-based commercial and in-house tests and the EUCAST-based zone edge test for penicillinase detection in S. aureus applying a PCR-based gold standard. Results In total, 215 non-duplicate clinical S. aureus isolates were included in the study, of which 127 (59.1%) did not harbour a blaZ gene, whereas 88 (40.9%) were blaZ positive. This study showed that for blaZ detection the zone edge test is more sensitive (96.6%) than nitrocefin tests independent of using nitrocefin discs (87.5% sensitivity) or solution (89.8% sensitivity), and that the significant inter-person variations of the zone edge test are probably related to the training level of the individual investigators (individual sensitivity ranging from 68.2% to 96.6%, specificity ranging from 89.8% to 100%). Conclusions In addition to continued and strict training of investigators, we propose mandatory checking of benzylpenicillin zone edges, particularly in an investigation zone from 26 to 30 mm, which can result in improved specificity/positive predictive value of the zone edge test (from 98.4% to 100%) but retains the high sensitivity/negative predictive value of the method.