Antiseptic agents are increasingly used for hand hygiene and skin decolonization as key tools for the prevention of healthcare-associated infections. Chlorhexidine, a divalent, cationic biguanide, has a broad spectrum of activity and is one of the most frequently used topical antiseptic agents. Notably, there are an increasing number of prevalence studies that report reduced levels of susceptibility to chlorhexidine. In contrast to bacterial resistance to antibiotics, using parameters such as the MIC to define resistance to antiseptics, including chlorhexidine, is not straightforward. A range of methods have been used for the detection of reduced susceptibility to chlorhexidine, but, importantly, there is no standardized method and no consensus on the definition of chlorhexidine 'resistance'. In this review we have assessed the methods available for the detection of reduced susceptibility to chlorhexidine and the prevalence of coresistance to other antimicrobial agents. We have focused on the development of reduced susceptibility to chlorhexidine and the presence of efflux-mediated resistance genes in staphylococci, and have reviewed the clinical significance of this phenomenon. Lastly, we have identified unanswered questions to further our understanding of this emergent threat. We anticipate that clinical use of chlorhexidine will continue to increase, and it will be important to be alert to the possibility that this may lead to the emergence of new clones with reduced susceptibility. Indiscriminate chlorhexidine use in the absence of efficacy data should be discouraged.