Phosphorus deficiency is severe in soil in south China. Symbiotic fungi plays an important role in plants uptaking phosphorus and other nutrients, such as mycorrhizal fungi. In this study, we sampled the roots of three main tree species in south China, Eucalyptus urophylla, Eucalyptus exserta, and E. urophylla × E. grandis. Root tissue separation was used to isolate and purify the fungi, which identified by morphological characteristics and ITS sequence analysis of 18S rDNA gene, and symbiotic fungi of Eucalyptus roots were validated by Kochs Rule. This study provides scientific evidence for theory research on symbiotic fungi in Eucalyptus roots and its resource utilization. The results showed that: (1) the collected roots of E. exserta displayed both ectomycorrhizas (ECM) and arbuscular mycorrhizas (AM) structures. Interestingly, both E. urophylla and E. urophylla × E. grandis possessed the structures of AM, ECM and dark septate endophytes (DSE). (2) Six fungi were isolated and identified from the roots of three species of Eucalyptus: Marasmius tricolor, Gymnopus melanopus, Phoma sp., Fusarium sp., Umbelopsis dimorpha, and Pestalotiopsis mangifolia, respectively. (3) Six fungi were inoculated back to roots of E. grandis tissue culture seedlings, M. tricolor and G. melanopus are able to form ECM structure in host roots as ECM fungi, whereas other four species of fungi (Phoma sp., Fusarium sp., U. dimorpha, and P. mangifolia) formed the dark color mycelium and microsclerotia in roots, the typical DSE structure, which were speculated to be DSE. The two ECM fungi were firstly reported in Eucalyptus roots.