Abstract When treated with various compounds, fungi exhibit characteristic morphological changes depending on their mode of action. Previously, we constructed morphology-based databases of the rice blast fungus Pyricularia oryzae and human pathogenic fungus Candida albicans and used them for antifungal screening. As these databases are manually created by human experts, objectivity and throughput may be a concern. To overcome these limitations, we developed a new artificial intelligence (AI)-based automated classification system for morphological changes in the filamentous fungus Aspergillus oryzae. Using this system, we screened a library of 7602 microbial broths and found that the culture broth of Streptomyces sp. RK21-A1205 exhibited potent antifungal activity and induced unique morphological changes distinct from those induced by conventional antifungal agents. Antifungal-activity-guided purification yielded the active metabolite RK-1205-I (1), whose chemical structure was identified as a new fostriecin derivative. 1 showed strong growth inhibitory activity against A. oryzae and other fungi, including A. fumigatus and C. auris, with IC50 value of 0.020 and 0.21 µM, respectively. Furthermore, 1 inhibited protein phosphatase 2A (PP2A) activity in the fungal cells. This study demonstrates that our newly developed AI-based phenotypic screening system can effectively distinguish morphological changes induced by microbial broths and facilitates the identification of novel antifungal compounds. This is a preview of subscription content, access via your institution Access options Subscribe to this journal Receive 12 print issues and online access $259.00 per year only $21.58 per issue Buy this article - Purchase on SpringerLink - Instant access to the full article PDF. USD 39.95 Prices may be subject to local taxes which are calculated during checkout Similar content being viewed by others References - Fisher MC, et al. Emerging fungal threats to animal, plant and ecosystem health. Nature. 2012;484:186–94. https://doi.org/10.1038/nature10947. - Sato T. Plant diseases and their pathogenic microbes in Japan. Microbiol Cult