The essential oil of the Melaleuca alternifolia (Maiden & Betche) (tea tree oil) has been effective in previous studies, in the treatment of infestation by Demodex mites in humans. The present study aimed at evaluating the in vitro acaricidal effect of this herbal medicine on Demodex canis. For the parasitological examination, samples were collected from a dog's skin and examined using optical microscopy. Only samples with intact mites and with evident movement of chelicerae and tarsi were selected. Twenty-one samples were tested with the oil, in seven different concentrations: 100%; 50%; 25%; 12.5%; 6.25%; 5.0% and 3.13%. Three samples were tested with the positive control amitraz, and three with the negative control neutral shampoo. The interval between the time the solution was added and the moment the movement of the last mite ceased defined the survival time in the sample. By comparing the times at different concentrations and controls, the results showed that the higher the concentration of the oil, the more quickly its lethal effect occurred, and that the survival times in the controls were longer than in the different concentrations of melaleuca oil.