Simple SummaryNosemosis in honeybees caused by theNosemaspp. infection is not only related to colony collapse but also increases the susceptibility of honeybees to various other pathogens; thus, it is necessary to develop effective control methods for nosemosis. Research on various control methods has been conducted, but clearly defined methods are still rare. In our study, the possibility of controlling honeybee nosemosis using entomopathogenic fungal culture extracts with various biological activities was evaluated. Many of the tested entomopathogenic fungal culture extracts showed high germination inhibitory activity onNosema ceranaespores. Among them, fungal culture extracts with high spore germination inhibitory activity and fungicidal activity was used to evaluate honeybee nosemosis inhibitory activity. As a result, it was confirmed for the first time that the fungal culture extract could not only suppress honeybee nosemosis but also increase the lifespan of honeybees. Entomopathogenic fungal culture extracts are expected to have applications in controlling honeybee nosemosis.AbstractThis study aimed to select the most effective culture extracts for controlling honeybee nosemosis using 342 entomopathogenic fungi of 24 species from 18 genera. The germination inhibitory activity of the fungal culture extract onNosema ceranaespores was evaluated using an in vitro germination assay method. Among 89 fungal culture extracts showing germination inhibitory activity of approximately 80% or more, 44 fungal culture extracts that maintained their inhibitory activity even at a concentration of 1% were selected. Finally, the honeybee nosemosis inhibitory activity was evaluated using the cultured extracts of five fungal isolates having aNosemainhibitory activity of approximately 60% or more, even when the extract was removed after treatment. As a result, the proliferation ofNosemaspores was reduced by all fungal culture extract treatments. However, only the treatment of the culture extracts fromPaecilomyces marquandii364 andPochonia bulbillosa60 showed a reduction in honeybee mortality due to nosemosis. In particular, the extracts of these two fungal isolates also increased the survival of honeybees.