Fusarium crown and root rot (FCRR) of tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici (FORL) is a common disease in worldwide. Our previous study showed the suppressive role of a dark septate endophyte Veonaeopsis simplex Y34 against Fusarium disease of Chinese cabbage, suggesting a potential bio-control agent to suppress FCRR of tomato. The aims of this study was to: 1) examine the suppressive potential of V. simplex Y34 against the FCRR of tomato; 2) explore the effect of V. simplex Y34 on root-endospheric community and how such effect links to the suppression of the disease. Two cultivars of tomato, House-momotaro and sicily-anrujo, were used in this experiment. The solid-cultures of V. simplex Y34, was applied in a nursery pot to assess the bio-control of the disease. Results showed that V. simplex Y34 decreased the disease severity of FCRR for the two cultivars. The colonization of V. simplex Y34 in root was determined by re-isolation and T-RFLP profiles targeting fungal ITS-LSU region. Moreover, the application of the endophyte increased the diversity and evenness of fungal community in root-endosphere and decreased the colonization of FORL in the root. NMDS analysis of T-RFLP profiles showed that the fungal communities in root-endosphere with inoculation of V. simplex Y34 were clustered away from those of non-inoculation, suggesting a correlation between the root-endospheric community and disease incidence. In conclusion, this study indicates that the application of V. simplex Y34 altered the diversity, evenness and structure of root-endospheric fungal community by the endosymbiosis of endophyte, and decreased pathogen colonization in the root, which opens up a new way to control of tomato FCRR disease. This work was supported by Council for Science, Technology and Innovation (CSTI), Cross-ministerial Strategic Innovation Promotion Program (SIP), “Technologies for creating next-generation agriculture, forestry and fisheries”.