The potential for emergence of resistance to biocides has been a concern in recent years. This study tested whether an association exists between the intensity of chlorhexidine use and chlorhexidine susceptibility of micro-organisms isolated from patients in different clinical areas in an acute-care general hospital. Organisms frequently involved in nosocomial infections in the hospital were chosen for study over a six week period: Staphylococcus aureus (60 isolates), coagulase-negative staphylococci (48), Klebsiella pneumoniae (32), Pseudomonas aeruginosa (60), Acinetobacter baumannii (16) and Candida albicans (35). An index of chlorhexidine exposure for each clinical unit was derived for the year preceding organism collection. Chlorhexidine susceptibility was evaluated using agar incorporation minimum inhibitory concentrations (MICs) and disk diffusion. A statistically significant inverse correlation was shown between intensity of chlorhexidine use and the overall susceptibility of all study organisms taken together. There was no association when individual taxa were considered. These findings must be interpreted with caution considering that greater use of chlorhexidine is likely to occur in difficult clinical disciplines where antibiotic use, invasive procedures and other intensive care-related procedures, cross-infection and immunosuppression are all potential confounding factors. There was an excellent correlation between MICs and disk testing, suggesting that disk diffusion might be useful in studies involving more than one biocide.