Fungicide applications in agriculture and medicine can promote the evolution of resistant, pathogenic fungi, which is a growing problem for disease management in both settings. Nonpathogenic mycobiota are also exposed to fungicides, may become tolerant, and could turn into agricultural or medical problems, for example, due to climate change or in immunocompromised individuals. However, quantitative data about fungicide sensitivity of environmental fungi is mostly lacking.Aureobasidiumspecies are widely distributed and frequently isolated yeast-like fungi. One species,A. pullulans, is used as a biocontrol agent, but is also encountered in clinical samples, regularly. Here, we compared 16 clinical and 30 agriculturalAureobasidiumisolates based on whole-genome data and by sensitivity testing with the 3 fungicides captan, cyprodinil, and difenoconazole. Our phylogenetic analyses determined that 7 of the 16 clinical isolates did not belong to the speciesA. pullulans. These isolates clustered with otherAureobasidiumspecies, includingA. melanogenum, a recently separated species that expresses virulence traits that are mostly lacking inA. pullulans. Interestingly, the clinicalAureobasidiumisolates were significantly more fungicide sensitive than many isolates from agricultural samples, which implies selection for fungicide tolerance of non-target fungi in agricultural ecosystems.IMPORTANCEEnvironmental microbiota are regularly found in clinical samples and can cause disease, in particular, in immunocompromised individuals. Organisms of the genusAureobasidiumbelonging to this group are highly abundant, and some species are even described as pathogens. ManyA. pullulansisolates from agricultural samples are tolerant to different fungicides, and it seems inevitable that such strains will eventually appear in the clinics. Selection for fungicide tolerance would be particularly worrisome for speciesA. melanogenum, which is also found in the environment and exhibits virulence traits. Based on our observation and the strains tested here, clinicalAureobasidiumisolates are still fungicide sensitive. We, therefore, suggest monitoring fungicide sensitivity in species, such asA. pullulansandA. melanogenum, and to consider the development of fungicide tolerance in the evaluation process of fungicides.