The activity of the antiseptic polyhexanide was tested against 250 gram-negative clinical isolates, that is, 50 isolates each of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Moraxella catarrhalis, and Haemophilus influenzae. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were determined by using a serial broth microdilution technique according to DIN 58940. Time-kill studies were performed for reference stains E. coli ATCC 25922, K. pneumoniae ATCC 4352, P. aeruginosa ATCC 15442, M. catarrhalis ATCC 43617, and H. influenzae ATCC 49247. All tested isolates had MICs and MBCs within a range of 1-32 mg/L and were regarded as susceptible to polyhexanide. The highest values were found for P. aeruginosa and H. influenzae with MICs and MBCs of 32 mg/L. Addition of up to 4% albumin to the test medium did not change MICs and MBCs. Time-kill studies of the reference strains showed reduction rates from 3 log10 colony forming units (CFU)/ml to more than 5 log10 CFU/ml for 200 and 400 mg/L polyhexanide within 5-30 min. Testing of polyhexanide in combination with antibiotics showed indifference with amoxicillin, cefotaxime, imipenem, gentamicin, and ciprofloxacin; no antagonism was found. As no resistance and no antagonism with antibiotics were detected, polyhexanide is regarded as suitable agent for topical eradication of gram-negative bacteria.