Background Healthcare-associated infection (HAI) is a major public health problem. As a form of prevention and control, preparations of chlorhexidine are used extensively; however, the reduction of susceptibility to chlorhexidine has been reported. The aim of this study was to investigate the susceptibility to chlorhexidine and the distribution of the qacA/B genes in 211 clinical isolates of coagulase-negative Staphylococci (CoNS). Methods CoNS were identified by conventional biochemical tests. Antimicrobial susceptibility was tested by disk-diffusion. Minimum inhibitory concentration (MIC) of chlorhexidine was determined by agar dilution test; detection of the qacA/B and mecA genes were evaluated by PCR. Results The most frequently isolated species were S. epidermidis , S. hominis hominis , S. auricularis, and S. haemolyticus , respectively. The strains presented a multidrug resistance profile of 87%, including methicillin resistance. Reduced susceptibility to chlorhexidine was observed in 31%. The qacA/B genes were detected in samples resistant (32/32) and susceptible (17/32) to chlorhexidine. The vast majority (94%) of the samples with reduced susceptibility to chlorhexidine were multidrug resistant. Conclusions Our results show that qacA/B genes are not restricted to strains expressing chlorhexidine resistance. Further studies are needed to understand how the expression of these genes occurs.