Lycium barbarum L. is an important economic crop in Qinghai province, China. However, root rot seriously reduces the economic results of L. barbarum. Here, we collected the diseased L. barbarum roots from Nuomuhong Farm of Haixi Mongolian and Tibetan Autonomous Prefecture, Qinghai Province, China, to clarify the diversity, pathogenicity, and biological characteristics of its root rot pathogens. A total of 125 isolates were collected, and based on morphological characteristics and rDNA ITS, TEF-1α, and RPB2 genes sequence analysis, they were identified as Fusarium equiseti, F. avenaceum, F. solani, F. citri, F. acuminatum, F. culmorum, F. sambucinum, F. incarnatum, F. oxysporum, F. tricinctum, Microdochium bolleyi, and Clonostachys rosea. These fungi were used to inoculate the roots of 1-year-old L. barbarum seedlings using scratching and root-irrigation inoculation methods, and all isolates caused root rot. This is the first report that M. bolleyi, F. avenaceum, and F. citri caused root rot in L. barbarum. And the best media, the lethal temperatures, and the optimum carbon sources and nitrogen sources of the 12 pathogen species were determined in this study. Moreover, our findings provide a theoretical foundation for root rot management in the future.