Entomogenous fungi have evolved many strategies for overcoming the defense systems that insects/mites, in turn, continue to evolve to fend off the fungi's attempt at infection. Although the host immune systems have garnered much attention, the importance of the cuticular defenses remains under-examined. The present study compared the mechanical properties – hardness and elastic modulus of the cuticles between a phytoseiid mite, Neoseiulus barkeri and a pest spider mite, Tetranychus urticae using nanoindentation techniques. We determined that the dorsal cuticle of N. barkeri possesses significantly greater hardness and stiffness, while the ventral cuticles of N. barkeri has mechanical properties similar to that of T. urticae. Exposure of the lateral abdomen region of N. barkeri after being distended by feeding, however, increased the susceptibility to fungal infection. It is found that the fungus can breach the exposed part of abdominal cuticle that is softer and less stiff, followed by conidial adhesion. This suggests mechanical properties of the dorsal cuticle of N. barkeri contribute substantially to withstand the mechanical force exerted by the attempted penetration of fungal hyphae. This study expand our knowledge of fungus–predator interactions and provide new insights into the feasibility of applying multiple biocontrol agents in pest control strategies.