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Previous studies have shown that foliar fungicides increase the abundance of Diaporthe spp. in mature soybean seeds, thereby reducing seed quality and potentially increasing seedling, stem, and pod disease in subsequent plantings. To mitigate this, we examined a fungicide containing mefentrifluconazole, fluxapyroxad, and pyraclostrobin applied during the R3 growth stage. We compared the seedborne fungal community from soybeans harvested at full maturity and 2 to 3 weeks later to assess fungal colonization. Six trials were conducted in Iowa and South Dakota in 2021 and 2022. Asymptomatic, recently harvested seed was assayed using either an agar-plate or a seed-blotter test, and the results were compared. The agar-plate method involved disinfesting seeds in sodium hypochlorite and ethanol and then transferring them to acidified water agar plates. Fungal isolates were identified using morphology and DNA sequences. This method detected 562 fungal isolates from 25 species and 14 genera. Dry conditions reduced seed infection, but a 2-week delay in harvest increased the likelihood of Alternaria and Cercospora infection in nontreated controls. The seed-blotter test involved disinfesting seeds, placing them on blotter paper, and incubating them. This assay detected higher numbers of fungal infected seed, with Aspergillus as the dominant genus. Storage conditions significantly impacted fungal presence, with more Aspergillus in seeds stored in a shed than in the cooler. Both assay methods showed that the fungicide treatment reduced the number of seeds infected with Alternaria, Cercospora, and Aspergillus. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.