Entomopathogenic fungi are facultative microorganisms, dwelling in soil or infecting host insects, and some of the genera have been used as biological control agents worldwide. Collection of fungal isolates should be a platform for the development of highly effective resources, thus in this work we constructed a fungal library using a mealworm (Tenebrio molitor) pathogenicity-based fungal collection method and further characterized some isolates with high virulence. A phylogenetic three was generated, and of the isolates 17 isolates’ biological features were characterized, such as morphology, spectrum of virulence, cultural characteristics, thermo-stability of fungi, production of biologically active materials, such as enzymes. This work reports an attractive entomopathogenic fungal library including the information of effective isolates in pest management.