Although the fungal microbiome in human skin mainly comprises lipophilic yeasts, including Malassezia species, these microorganisms can cause various dermatitis conditions, including pityriasis versicolor, seborrheic dermatitis, folliculitis, and atopic dermatitis, depending on the host condition. Both Malassezia globosa and Malassezia restricta are major species implicated in Malassezia-related dermatitis. However, the pathogenicity of these microorganisms has not been revealed at the genetic level owing to the lack of a genetic recombination system. Therefore, we developed a gene recombination system for M. globosa using Agrobacterium tumefaciens-mediated gene transfer of the target gene FKB1, which encodes the FKBP12 protein that binds the calcineurin inhibitor tacrolimus. The wild-type strain of M. globosa was sensitive to tacrolimus, whereas the FKB1 deletion mutant was resistant to tacrolimus. Reintroduction of FKB1 into the FKB1 deletion mutant restored wild-type levels of susceptibility to tacrolimus. Moreover, an FKB1-eGFP fusion gene was generated and expression of this fusion protein was observed in the cytoplasm. This newly developed gene recombination system for M. globosa will help further our understanding of the pathogenesis of M. globosa-related dermatitis at the genetic level.