Showdomycin is a potent nucleoside antibiotic that displays a high structural similarity to uridine and pseudouridine. No detailed target analysis of this very unusual electrophilic natural product has been carried out so far. To unravel its biological function, we synthesized a showdomycin probe that can be appended with a fluorophor or a biotin marker via click chemistry and identified diverse enzymes which were important for either the viability or virulence of pathogenic bacteria. Our results indicate that the antibiotic effect of showdomycin against Staphylococcus aureus may be due to the inhibition of various essential enzymes, especially MurA1 and MurA2, which are required for cell wall biosynthesis. Although real-time polymerase chain reaction revealed that the MurA2 gene was expressed equally in four S. aureus strains, our probe studies showed that MurA2 was activated in only one multiresistant S. aureus strain, and only this strain was resistant to elevated concentrations of the MurA inhibitor fosfomycin, suggesting its potential role as an antibiotic bypass mechanism in the case of MurA1 inhibition. Moreover, we utilized this tool to compare enzyme profiles of different pathogenic strains, which provided unique insights in regulatory differences as well as strain-specific signatures.