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Potatoes rank third in terms of human consumption after rice and wheat. <i>Globodera</i> spp. are significant pests of potato crop worldwide. <i>Globodera rostochiensis</i> was found in Weining County, Guizhou Province, China, in 2019. We collected soil from the rhizosphere zone from infected potato plants and separated mature cysts through simple floatation and sieving methods. The selected cysts were surface-sterilized, and the colonized fungi were isolated and purified. At the same time, the preliminary identification of fungi and fungi parasites on the cysts of nematodes was carried out. This study aimed to define the species and frequency of fungi-colonizing cysts of <i>G. rostochiensis</i> collected from Weining County, Guizhou Province, China, and provide a basis for the control of <i>G. rostochiensis</i>. As a result, 139 strains of colonized fungi were successfully isolated. Multigene analyses showed that these isolates included 11 orders, 17 families, and 23 genera. The genera <i>Fusarium</i> (with a separation frequency of 59%), <i>Penicillium</i> (11%), <i>Edenia</i> (3.6%), and <i>Paraphaeosphaeria</i> (3.6%) were the most frequently occurring. Among the 44 strains, 27 had a colonization rate of 100% on the cysts of <i>G. rostochiensis</i>. Meanwhile, the functional annotation of 23 genera indicated that some fungi have multitrophic lifestyles combining endophytic, pathogenic, and saprophytic behavior. In conclusion, this study showed the species composition and lifestyle diversity of colonized fungi from <i>G. rostochiensis</i> and demonstrated these isolates as potential sources of biocontrol agents. Colonized fungi were isolated from <i>G. rostochiensis</i> for the first time in China, and the taxonomic diversity of fungi from <i>G. rostochiensis</i> was clarified.