Background -Approved treatments for canine otitis externa are limited in variety and may contain ototoxic ingredients. With bacterial resistance an ongoing concern, it would be ideal if non-ototoxic agents combined with antibiotics resulted in a synergistic effect, requiring lower antibiotic concentrations to treat infections. Evidence of synergism and antagonism between N-acetylcysteine (NAC) and various antibiotic classes has been reported; the present research group was interested in examining these interactions.Hypothesis/Objectives -To determine if NAC, an otoprotective and antimicrobial compound, has synergistic activity when combined with enrofloxacin or gentamicin in vitro against bacterial isolates causing canine otitis externa.Animals -Twenty-two isolates from canine clinical cases of otitis externa were identified and tested, including seven Staphylococcus pseudintermedius, 12 Pseudomonas aeruginosa and three Corynebacterium spp. isolates.Methods and materials -Each isolate was grown on blood agar for 24 h and transferred to Mueller-Hinton broth (MHB), with a final concentration of 5 9 10 5 cfu/mL. Each well was inoculated with 50 lL of bacterial suspension. N-acetylcysteine was diluted in MHB to a starting concentration of 160 mg/mL. Enrofloxacin and gentamicin were diluted to 64 lg/mL. Individual and checkerboard serial microdilution assays were performed in triplicate with negative controls for all isolates tested.Results -Interactions observed for NAC and enrofloxacin were synergistic (4.5%), indifferent (77.3%) or antagonistic (18.2%). Interactions observed for NAC and gentamicin were synergistic (4.5%), indifferent (45.5%) or antagonistic (50%).Conclusions and Clinical relevance -Most interactions between NAC and enrofloxacin or gentamicin were indifferent or antagonistic at the concentrations tested in vitro.