Bacterial biofilms can complicate the treatment of infections because bacteria within the biofilm may evade desiccation, host immune response, and antimicrobial therapy. Recently, biofilm production has been documented in canine isolates of Pseudomonas aeruginosa. In this study, 31 canine otic isolates of multidrug-resistant P aeruginosa previously shown to produce a biofilm were tested to determine whether Tris-buffered EDTA had an impact on their antimicrobial susceptibility. The minimum inhibitory concentration (MIC) of neomycin, polymyxin B, enrofloxacin, and gentamicin were determined for biofilm-embedded bacteria with added Tris-buffered EDTA. The MIC of neomycin and gentamicin decreased with the addition of Tris-buffered EDTA, but not for polymyxin B. The MIC of enrofloxacin increased in the presence of Tris-buffered EDTA.