Background Canine generalized demodicosis is a common parasitic disease caused by the proliferation of Demodex mites. The introduction of isoxazoline class treatments in veterinary dermatology has resulted in apparently effective treatment of generalized demodicosis. The objective of this study was to evaluate the effectiveness of fluralaner for the treatment of canine generalized demodicosis using real-time PCR for the detection and quantification of Demodex DNA. Methods Twenty privately owned dogs with clinical symptoms of generalized demodicosis and deep skin scrapings positive for Demodex canis mites were enrolled in the study. Following diagnosis (day 0) each dog was treated with fluralaner at the recommended commercial dose for tick and flea treatment (25–56 mg/kg) based on body weight. Clinical and mite count assessments, and hair sampling for molecular analyses were performed on days 0, 28, 56, 84 and 112. Demodex DNA was detected and quantified using real-time PCR. Results A single oral dose of fluralaner reduced Demodex mite counts in skin scrapings by an average of 98.9% in all dogs by day 28. No mites were recovered from skin scrapings from any treated dog by day 56, at which time the dog was considered to be clinically cured, with total hair regrowth. There were significant differences among examined dogs in qPCR cycle threshold (Ct) values on days 0, 28, 56, 84 and 112. Demodex DNA levels decreased (increasing Ct values) throughout the study. Mite DNA was present on day 112, possibly from dead mites, at values significantly lower than in samples taken on days 0, 28 and 56. Based on qPCR testing of diluted samples, the Demodex mite population was reduced by approximately 1000-fold on day 112. Conclusions Oral administration of fluralaner at the recommended dose to dogs with generalized demodicosis is highly effective for reducing Demodex mite populations and resolving clinical signs of generalized demodicosis. The presence of mite DNA may indicate that treatment did not kill all Demodex mites.