BackgroundOtitis externa (OE) is one of the most frequently diagnosed dermatological diseases in dogs, having a multifactorial aetiology. Among the bacterial agents associated with canine OE, Pseudomonas aeruginosa is of special concern owing to its frequent multidrug resistance profile and ability to form biofilms related to the infection's chronicity and recurrence.ObjectivesThe main objective of this study was to evaluate and compare the antibiofilm activity of two innovative antimicrobials—an otological gel containing a synthetic antimicrobial peptide and Lavandula angustifolia essential oil—with gentamicin (a conventional antibiotic) using biofilm‐producing P. aeruginosa isolates obtained from dogs with OE.Materials and MethodsThe biofilm eradication capacity of gentamicin, otological gel and lavender oil was determined against a collection of 12 P. aeruginosa biofilm‐producers among 35 clinical isolates obtained from the ear canals of dogs with OE. Also, the antimicrobial activity of the otological gel against P. aeruginosa biofilms was assessed in an in vitro model of dog cerumen.ResultsLavender oil showed the best effectiveness after 30 min of contact, eradicating 58.3% (seven of 12) of the isolates, and gentamicin showed full eradication (12 of 12) after 24 h. The otological gel acted more slowly than the lavender oil; yet at 24 h, the antibiofilm capacity of both compounds was similar, with no significant difference between them. It also was found that triglycerides from synthetic cerumen earwax had antipseudomonal activity and, when combined with the otological gel, led to the full eradication of P. aeruginosa.Conclusions and Clinical RelevanceThe results of this in vitro study indicate that lavender oil and the otological gel may be effective topical treatments for canine OE promoted by P. aeruginosa biofilm‐producers, as alternatives to gentamicin.