Glycosidases fulfill crucial functions in all life forms contributing to diverse biological processes such as metabolism, cell signaling, or pathogen-host interactions. In search for new putative targets for antimicrobial drugs, we here aimed to identify and characterize retaining glycosidase activities in the bacterial pathogen Staphylococcus aureus. We initially screened the activities of diverse retaining glycosidase families in S. aureus JE2 under different growth conditions using panel of seven different fluorescent activity-based probes in a gel-based system, before using a pool of three related biotinylated probes for target enrichment and identification by mass-spectrometry-based activity-based protein profiling. Three glycoside hydrolase family proteins were identified: 6-phospho-β-glucosidase (BglA), α-amylase family protein trehalase C (TreC), and autolysin (Atl). The physiological relevance of the previously uncharacterized glycosidases BglA and TreC were addressed using CRISPRi-induced gene silencing. Whereas both enzymes were dispensable for in vitro growth in Tryptic Soy Broth, silencing of treC, but not bglA, led to reduced virulence in a Galleria mellonella infection model, warranting future investigations into the biochemical function and physiological role of this enzyme.