Minimum bactericidal concentrations (MBCs) of a commercial ear antiseptic containing chlorhexidine 0.15% and Tris-EDTA (Otodine) were determined by broth microdilution for 150 isolates representing the most common pathogens associated with canine otitis. The microorganisms were classified into three groups according to their levels of susceptibility. The most susceptible group included Staphylococcus pseudintermedius, Malassezia pachydermatis, Streptococcus canis and Corynebacterium auriscanis, which were generally killed by 1 : 64 dilution of the antiseptic product (MBC = 23/0.8 microg/mL of chlorhexidine/Tris-EDTA). The most resistant organism was Proteus mirabilis, which survived up to 1 : 8 dilution of the product (MBC = 375/12 microg/mL). Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus displayed intermediate MBCs ranging between 188/6 and 47/1.5 microg/mL. Interestingly, S. pseudintermedius was more susceptible than S. aureus, and no significant difference was observed between meticillin-resistant and meticillin-susceptible isolates within each species, indicating that antiseptic use is unlikely to co-select for meticillin resistance. Although the concentrations required for killing (MBCs) varied considerably with microorganism type, the combination of chlorhexidine 0.15% and Tris-EDTA was active against all the pathogens most commonly involved in canine otitis.