The purpose of this study was to evaluate in detail both the in vivo and in vitro efficacy of the enzyme agents, ZYMOX® Plus Otic (ZYMOX-P), in the treatment of canine otitis externa (OE). Eight dogs with a diagnosis of non-seasonal severe chronic OE were recruited for the study. ZYMOX-P was administered for 2-4 weeks. The Otitis Index Score (OTIS3) and bacteria or yeast colony growth were measured. Also, minimum biofilm (BF) formation inhibition concentration (MBIC) and BF bactericidal concentration (BBC) were measured in vitro. OTIS3 showed a statistically significant reduction after treatment (88.2%, p⟨0.001; pre-treatment = 11.0 ± 0.9; post-treatment = 1.3 ± 0.4, mean ± SEM). The individual OTIS scores, erythema, edema, erosions/ ulcerations, exudate and pruritus showed significant reduction (85.7%, 95.7%, 83.3%, 80.0%, and 89.3%, respectively). Microscopic examination revealed the presence of BF exopolysaccharide in all 8 ear samples when stained with alcian blue. Seven of the 8 dogs (87.5%) showed a reduction in colony growth. ZYMOX-P was effective at 34-fold and 16-fold dilutions on MBIC and BBC, respectively. These findings indicate that ZYMOX-P has efficacy against BF-related infection and is beneficial when used for the management of canine OE.