Background This study aimed to evaluate lotilaner (Credeli®)’s larvicidal efficacy in the treatment of dogs naturally infested with Dermatobia hominis larvae.Methods A total of 12 dogs that naturally presented at least three live D. hominis larvae were medicated. All the animals could not have undergone any type of insecticide or acaricide and/or larvicidal treatment in a period before 90 days, as an inclusion criterion. The animals were medicated with a single dose of at least 20 mg/kg lotilaner in the oral formulation, following the recommendations in the package insert for other ectoparasitic treatments. After drug administration, the animals remained at their homes, and observations were made to verify the larvicidal effect 6 h after treatment, in addition to possible adverse effects on the medicated animals. Live larvae were considered as any parasite that showed motility after removal. The effect was calculated for each animal using the formula: [100 × (number of live larvae before treatment − number of live larvae after treatment/number of live larvae before treatment)] as criteria for evaluating lotilaner efficacy.Results A total of 98 larvae were counted in 12 dogs, with an average of 8.1 larvae per animal. The majority of the parasites, that is, 38 larvae, were found in the animals’ dorsal region. Lotilaner’s effectiveness was 80.6%. No animal showed adverse effects or changes in behavior after isoxazoline use. Nineteen larvae were found alive, presenting hypomobility and lethargic behavior. Notably, the evaluation only lasted for 6 h.Conclusion Lotilaner, when orally administered in a single dose of 20 mg/kg, is effective in the treatment of dogs naturally infested by D. hominis.