The yeastMalassezia pachydermatisis a common inhabitant of the skin and mucosae of dogs. However, under certain circumstances this yeast can overgrow and act as an opportunistic pathogen causing otitis and dermatitis in dogs. Canine pododermatitis is a common disorder in dogs in whichM. pachydermatisacts as an opportunistic pathogen. In the present study, the presence ofMalasseziayeasts was assessed and quantified in samples collected from the interdigital space of dogs with pododermatitis before and after treatment, and from healthy dogs. The samples were subjected to two different cytological examinations, culture on Sabouraud glucose agar and modified Dixon’s agar and a quantitative PCR targeting the internal transcribed spacer (ITS) genomic region. A selection of samples was analyzed by next generation sequencing (NGS) using the D1D2 domain of the large subunit of the ribosomal DNA as target. The pododermatitis samples before treatment showed higher cell counts, colony-forming units and ITS copies than the rest of samples. The NGS analysis revealed thatAscomycotawas the main phylum in the healthy and post-treatment samples. However,BasidiomycotaandM. pachydermatiswas more abundant in the pododermatitis samples before treatment. These results supportM. pachydermatisas an opportunistic agent in canine pododermatitis by a variety of methods, and demonstrate the correlation between cytologic and molecular methods for quantification.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11259-022-09951-2.