Lufenuron is a benzyl-urea phenol compound that inhibits chitin synthesis and is used as an insecticide. Its efficacy in the therapy of dermatophytosis in dogs and cats was evaluated in several clinical studies, with contradictory results. We assessed the in vitro susceptibility of dermatophytes isolated from dogs and cats to lufenuron, and the clinical response of skin lesions to the drug. Dermatophyte cultures isolated from clinical cases were exposed to lufenuron by three different methods: direct application and application of whole blood or subcutaneous tissue samples obtained from a lufenuron-treated healthy dog. No inhibition of dermatophyte growth was observed in any of the samples after 1 week of incubation. Eight dogs and six cats with skin lesions were included in the in vivo survey. Results indicated that six of seven skin lesions that were diagnosed as being caused by dermatophytes did not respond to lufenuron whereas six of seven skin lesions that were not caused by dermatophytes improved. We concluded that lufenuron, in the way it was administered in this study, had no inhibitory activity on dermatophytes in vitro or in vivo and its clinical use as an anti-fungal agent is questionable. An immunomodulatory effect of the drug is, however, possible.