The membrane-active antimicrobial agent chlorhexidine is used extensively as an antiseptic during infection prophylaxis and treatment. Whilst known to induce membrane damage that results in loss of internal solutes from bacteria, the present study sought to determine the rate and extent of cytoplasmic potassium loss and whether any species-specific differences exist. Direct measurement of potassium was achieved using flame emission spectrophotometry. Exposure of selected species to minimum inhibitory (MIC) or minimum bactericidal concentration (MBC) resulted in solute loss that was both concentration and time dependent. Within 5-min treatment with MIC levels, losses of 3 % from P. aeruginosa, 9 % from E. coli, and 15 % from S. aureus were recorded, whilst at 5 % w/v chlorhexidine, elevated loss of 20, 28, and 41 % occurred, respectively. Nonlinear potassium release was evident from all species when treated with 5 % chlorhexidine over a 60-min period. After this contact time, potassium loss from E. coli and S. aureus rose to 93 or 90 %, respectively; in contrast, P. aeruginosa retained 62 % intracellular potassium. Results confirm lethal concentrations of chlorhexidine induce rapid and substantial loss of cytoplasmic potassium from common pathogens. However, bacterial responses vary between species and should be borne in mind when considering mechanism of action.