Compared with traditional biological control, the co-use of entomopathogenic fungi and multiple enemies has made great progress in biocontrol technology. However, the risk posed by entomopathogenic fungi to their host has not been fully evaluated. Further, the interaction between them has not described adequately. In this study, we investigated the crucial role of peptidoglycan recognition protein (PGRP), an important pattern recognition receptor, involved in the resistance of Neoseiulus barkeri against the entomopathogenic fungus, Beauveria bassiana. Results showed that N. barkeri possessed one NbPGRP gene, which was mainly enriched in the ventral cuticle, and was significantly upregulated after B. bassiana induction. Knocking down NbPGRP resulted in decreased survival rate of mites and increased B. bassiana load. Also, recombinant NbPGRP inhibited the proliferation of B. bassiana, but also attached to spores by binding to surface PAMPs. Furthermore, the transcriptomics after NbPGRP silencing indicates that NbPGRP has a potential regulatory role in mite physiologies. These results showed that NbPGRP has a dual function of inhibiting the proliferation of entomopathogenic fungi and regulating mite resistance upon recognition of entomopathogens. This enhances understanding of the interaction mechanism between entomopathogens and their insect hosts.