Activity-based protein profiling is a powerful chemoproteomic technique to detect active enzymes and identify targets and off-targets of drugs. Here, we report on the use of a carmofur-based activity-based probe to identify biologically relevant enzymes in the bacterial pathogen Staphylococcus aureus. Carmofur is an anti-neoplastic prodrug with antimicrobial activity that is related to 5-fluorouracil. The carmofur-probe was originally designed to target human acid ceramidase, a member of the Ntn hydrolase family with an active-site cysteine nucleophile. Here, we first profiled the targets of a fluorescent carmofur-probe in live S. aureus under biofilm-promoting conditions and in liquid culture, before proceeding to target identification by liquid chromatograpy/mass spectrometry. Treatment with a carmofur-biotin probe led to enrichment of 21 enzymes from diverse families awaiting further characterization, including the Ntn hydrolase-related IMP cyclohydrolase PurH. However, the probe preferentially labelled serine hydrolases, thus displaying a reactivity profile similar to carbamates. Our results suggest that the electrophilic N-carbamoyl-5-fluorouracil scaffold could potentially be optimized to achieve selectivity towards diverse enzyme families. The observed promiscuous reactivity profile suggests that the clinical use of carmofur presumably leads to inactivation of a number human and microbial enzymes that could lead to side-effects or even contribute to therapeutic efficacy.