Several microorganisms as bacteria, fungi, yeast and ectoparasites compose canine ear canal microbiota. These agents can be native or invasive pathogens that cause clinical otitis. The aim of this study was to associate Mollicutes in external ear canal with clinical otitis. 41 domestic dogs were examined, and a total of 82 otologic samples were collected using sterile swabs, placed in a plastic storage tube containing 2 milliliters of sterile phosphate buffer saline (PBS, pH 7.2) and appropriately labeled. The samples were processed for isolation of Mollicutes using Hayflick’s medium. The frequency of Mollicutes class in the isolation was 32.9% (27/82). 14.8% (4/27) of the dogs, which were positive for Mollicutes, had external otitis and 85.2% (23/27) were healthy dogs. The genus Mycoplasma spp. was detected in 7.4% (2/27) by digitonin test. It was the first isolation of Mollicutes from the external ear canal in dogs. Although there have been important findings in this study, the presence of these bacteria cannot indicate the onset and perpetuation of external otitis in dogs.