Malassezia pachydermatisis part of the normal skin microbiota of various animal species but under certain circumstances becomes an opportunistic pathogen producing otitis and dermatitis. Commonly theseMalasseziadiseases are effectively treated using azoles. However, some cases of treatment failure have been reported. Alterations in theERG11gene have been associated with in vitro azole resistance inM. pachydermatis. In the present study, in vitro antifungal susceptibility of 89 different strains ofM. pachydermatisisolated from different animal species and health status was studied. The susceptibility to fluconazole (FLZ), itraconazole (ITZ), ketoconazole and amphotericin B was tested by a disk diffusion method and 17 strains were also subjected to an ITZ E-test. Mueller–Hinton supplemented with 2% glucose and methylene blue was used as culture medium in both susceptibility assays. Multilocus sequence typing was performed in 30 selected strains using D1D2, ITS, CHS2 and β-tubulin genes. Also,ERG11gene was sequenced. The four antifungals tested were highly effective against most of the strains. Only two strains showed no inhibition zone to antifungals and a strain showed an increased MIC to ITZ. The study of theERG11sequences revealed a high diversity of DNA sequences and a total of 23 amino acid substitutions, from which only two have been previously described. Also, three deleterious substitutions (A302T, G459D and G461D) previously associated with azole resistance in this yeast were recovered. A correlation between certain genotypes andERG11mutations was observed. Some of theERG11mutations recovered were correlated with a reduced susceptibility to azoles.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11046-022-00696-9.