Honeybees' gut microbiota can provide new valuable access into the pathogenesis-related factors included in infections. Hence, we researched the presence and comparison of gut microbiota groups in control and spp.-infected honeybee colonies through high-throughput sequencing of the 16S rRNA. As the newest approach in apiary management, we hypothesize that the EM probiotic for bees could have an important role in therapeutic and immunomodulatory effects on honeybee colonies. The aim of this study was to estimate its impact on the gut microbiota composition of adult honeybees. The major genera were detected, where Lactobacillus was the most abundant genus, followed by Gilliamela, Snodgrassella, and Bifidobacterium. Inoculation with spp. spores made the relative proportions of Bifidobacterium lower, which was ameliorated by EM for bees' application. In addition, EM for bee applied treatments suppressed the increase in the number of spp. spores. This result points out that continuous EM for bees treatment shall change bees' gut microbiome composition and mitigate the influence of spp. infection. was a major member of the honeybee gut microbiota and may be significantly increased by long-term treatment with EM for bees. Toward these results, it is possible that EM for bees treatment will protect honeybees from herbicide glyphosate negative effects in agricultural fields by improving microbiome and immune functions.