In order to correctly identify a new biovar of Staphylococcus aureus subsp, aureus, (NBSA) a simple, rapid, and reliable enzymatic assay was developed. The assay was based on the detection of the production of three enzymes: alpha-glucosidase, beta-glucosidase and beta-N-acetyl-glucosaminidase. Of a total of 46 isolates of Staphylococcus aureus subsp. aureus from clinical specimens, the new assay correctly identified 19 as NBSA and 27 as typical Staphylococcus aureus subsp. aureus. Among the 19 NBSA isolates, 15 (79%) showed a clear biochemical profile while only four isolates (21%) showed a less well-defined enzymatic combination, due to a phenotypic alteration caused by subculturing. Since this assay is both simple to perform and inexpensive, it is potentially applicable in the laboratory.