Enterococcus faecium has emerged as a major nosocomial and opportunistic pathogen, increasingly associated with severe infections such as urinary tract infections, bacteraemia, and endocarditis. Notably, it exhibits intrinsic resistance to many antibiotics and has a remarkable capacity to acquire additional resistance mechanisms, making it particularly difficult to treat—especially due to its heightened resistance to vancomycin and ampicillin. To address these therapeutic challenges, we employed a chemical proteomics strategy to investigate ATP-binding protein activity in E. faecium. Using the activity-based probe (ABP) desthiobiotin-ATP, we profiled protein activity under different growth conditions, including Brain-Heart Infusion (BHI) broth supplemented with either human serum or colon organoid extract. Our analysis identified 230 enriched putative ATP-binding proteins. Of these, 31 proteins were specifically activated in response to the organoid extract, and 19 in response to human serum. Notably, the ABC transporter TcyA and the histidine kinase ArlS were activated in the presence of the organoid extract. These findings suggest that E. faecium dynamically modulates its metabolic and regulatory pathways in response to host-derived signals from human serum and organoid effluent, potentially enhancing its adaptability and pathogenicity in various host environments. We highlight TcyA and ArlS for their known roles in virulence in other pathogens, making them promising targets for further functional studies in E. faecium.