This study utilized next-generation sequencing to describe the natural fungal microbiome (mycobiome) of canine skin and to identify changes in the mycobiome when allergic skin disease is present. Superficial skin swabs from ten dogs with healthy skin and eight dogs with allergic skin disease were collected from haired and non-haired sites, 10 sites for healthy dogs and 6 sites for allergic dogs. The DNA was extracted from skin swabs and the Internal Transcribed Spacer (ITS) region was sequenced using an Illumina Miseq instrument. The total number of ITS sequences for 144 samples was 5,604,145, with the median number of sequences per sample being 38,680. Sequences were analyzed using the QIIME platform. We identified the most prevalent fungal genera as Cladosporium and Alternaria. Other significant genera identified included Malassezia, Fusarium, Cryptococcus, Claviceps, Wallemia, Kodamaea, Podosphaera, and Blumeria. In the healthy dogs, the greatest diversity (number of observed species) in fungal genera was found on the dorsal aspect of the nose, while the least diverse sites included the nostril and conjunctiva (p