Chronic otitis externa in dogs is often associated with Pseudomonas aeruginosa infection. Fluoroquinolones are often used for treating such infections. Fluoroquinolone resistance mechanisms were characterized in 10 strains of P. aeruginosa isolated from chronic canine otitis externa. Nine out of ten strains harbored a mutation in the gyrA gene and presented an overexpression of efflux pump(s). There was a good correlation between the lipophilicity of the fluoroquinolone being tested and the effect of the efflux pump inhibitor in the final MIC. Therefore, both mechanisms, mutation in the gyrA gene and increased efflux pump(s), seem to play an important role in the acquisition of fluoroquinolone resistance in veterinary clinical isolates of P. aeruginosa. Levels of resistance to fluoroquinolones suggest that they could not be a good choice for systemic therapy of Pseudomonas otitis.