BACKGROUND: Onchocerca lupi and Cercopithifilaria spp. are vector-borne filarioids of dogs, which harbour skin microfilariae (mfs), the former being of zoonotic concern. Proper treatment studies using compounds with microfilaricidal activity have not been performed. Therefore, this study aimed to assess the efficacy of a commercially available spot-on formulation containing moxidectin 2.5%/imidacloprid 10% for the treatment of O. lupi or Cercopithifilaria spp. skin-dwelling mfs in naturally infected dogs. METHODS: Privately owned dogs (n = 393) from southern Portugal were sampled via skin biopsies to identify and count mfs in 20 µl of skin sediment. A total of 22 mfs-positive dogs were allocated to treatment group (n = 11; G1) or left untreated as a control (n = 11; G2). As a pilot investigation to test the treatment efficacy, five dogs assigned to G1 were treated four times at monthly intervals with moxidectin 2.5%/imidacloprid 10% spot-on formulation on SDs 0, 28 (± 2), 56 (± 2), and 84 (± 2). Based on the negative results for both O. lupi and/or Cercopithifilaria spp. mfs of dogs in the pilot study from SD28 onwards, the remaining six dogs in G1 were treated at SD0 and assessed only at SD28. RESULTS: Of the 393 animals sampled, 78 (19.8%) scored positive for skin-dwelling mfs. At the pilot investigation, a mean number of 19.6 mfs for O. lupi was recorded among five infected dogs whereas no mfs were detected at SD28. At SD0, the mean number of Cercopithifilaria spp. larvae was 12.6 for G1 and 8.7 for G2. The mean number of mfs for G2 was 20.09. CONCLUSIONS: Results herein obtained suggest that a single treatment with moxidectin 2.5%/imidacloprid 10% spot-on formulation is efficacious against skin-dwelling mfs in dogs. The microfilaricidal effect of moxidectin could also be useful in reducing the risk of O. lupi infection for humans.