Two-spotted spider mite (Tetranychus urticae Koch; TSSM) is an agriculturally serious pest tat has acquired strong resistance against acaricide. Alternatively, mite-pathogenic fungi could be used to control the mites. The spider mite has symbiotic microorganisms which could be involved in the physiological and ecological adaption to biotic stress. In this study, mite-pathogenic fungi were used to control female adults, and the change of microbiome in the fungus-infected mites was analyzed. The acaricidal activity of fungal isolates was tested, and Akanthomyces attenuatus JEF-147 with the highest acaricidal activity was determined, followed by semi-field assay. Microbiome in the female adults which was infected by A. attenuatus JEF-147 was analyzed, and composition of microorganism was changed by fungal treatment. In bacteria abundance, the arthropod defense-related Rickettsia increased, but reproduction-associated Wolbachia decreased. The change in major bacterial abundance in the infected mites could be explained by the trade-off between reproduction and immunity. This work describes that a possible trade-off in arthropods against fungal pathogens could be predicted by the microbiome analysis.