Entomopathogenic fungi (EF), who represent active agents to control insect natural populations, usually persist in terrestrial habitats. Southwest area in China has various climate conditions and abundant plant biodiversity (crop, forest, grassy, orchard and arable areas). Nevertheless, the potential of soil-inhabitant EF as insect pest biocontrol agents, is unknown. In this study, first the EF biodiversity from soil of three provinces (Guizhou, Sichuan, and Yunnan) was surveyed. Then, the virulence of 29 isolated strains against Bemesia tabaci and Spodoptera litura was assessed. After analyzing 212 soil samples, 497 isolated fungi were identified. Out of them, 490 isolates were classified in 45 species of 24 genera, whereas the other seven isolates, belonging to Paecilomyces and Purpureocillium genera, were not identified under species level. Furthermore, the EF biodiversity from soil of Sichuan, Yunan, and Guizhou areas, analyzed by Shannon Wiener Index (SWI) was rated at 2.98, 1.89, and 2.14, while SWIs-biodiversity in crop, forest, grassy, orchard and arable areas was rated at 2.88, 2.74, 3.05, 2.39, and 2.47. SWI data suggested that soil from Sichuan area and grassy had higher EF biodiversity compared with other analyzed provinces and areas. Virulence bioassay results indicated that, out of the 29 isolates tested, 24 were pathogenic against B. tabaci and S. litura, resulting in mortality rates >10%. In conclusion, this study reports the EF distribution and biodiversity in soil from three provinces located at Southwest China, whereas their potential use as a tool for the B. tabaci and S. litura biocontrol must be further investigated.