This study evaluated the efficacy of a new, single-dose otic solution combining florfenicol, terbinafine, and mometasone furoate for the treatment of canine otitis externa (OE) in vitro and in vivo. Forty-one client-owned dogs with OE were included in the study and divided into a treatment group that received the test solution and a negative control group that received a normal saline solution. On day 0, the dogs were treated either with the test or the control solution and evaluated over 30 days. Clinical efficacy was evaluated by clinical signs and cytological organism counts. In vitro antimicrobial susceptibility was evaluated by the minimum inhibitory concentration (MIC). After treatment with the test solution, clinical signs continuously decreased and cytological scores were significantly reduced. The results of MIC testing showed that the test solution was potent against the common pathogenic causes of canine OE. In this study, the most common causative pathogens were Staphylococcus pseudintermedius, Pseudomonas spp. and Malassezia pachydermatis. No issues related to safety were identified. Based on these results, this new ototopical drug can be used as first line treatment for canine OE.