Enterococcus faecium, a human and animal commensal broadly distributed in the environment, is currently one of the most challenging multidrug-resistant (MDR) healthcare-associated pathogens worldwide. It is often exposed to chlorhexidine (CHX), a broad-spectrum antiseptic, extensively used in healthcare, domestic, and food production settings, and a diffused polluter. However, the impact of gradients of CHX concentrations, including at subinhibitory levels, on E. faecium adaptation to various antimicrobials remains unclear. Our study aimed to explore the effects of subinhibitory CHX concentrations on biocides and antibiotics susceptibility as well as in the transfer of clinically relevant antibiotic resistance genes among E. faecium (n = 11) from diverse sources and clonal backgrounds. Serial exposure to increasing CHX concentrations resulted in strain-specific MIC