To study the effects of mannan oligosaccharides (MOS) on rumen fungal flora of dairy cows, four lactating Chinese Holstein dairy cows were randomly divided into two groups in the two-stage 2 × 2 cross-over design. The experimental group (MT) was fed with a basal ration and MOS were perfused orally 60 g/(d head). The control group (CK) was fed with a basal ration. The rumen fluid was collected through the oral cavity of dairy cows using a negative pressure suction device. 50ml rumen fluid of every cow was collected each time respectively before feeding (0 h) and 2h, 4h and 6h after feeding in a day. The rumen fungi were tested by ITS sequencing technology. The results showed that four phyla were identified in CK group and five in MT group. The dominant phyla in both groups were Ascomycota, Basidiomycota, Neocallimastigomycota, whose abundances were more than 90% of the total rumen fungi. At the level of genus, 74 genera were identified in CK group, while 91 genera in MT group. Several cellulolytic fungi genera showed a decreasing trend or decreased significantly, such as Neocallimastigaceae-NA. The previous experiments that shows the addition of mannan oligosaccharides promoted the proliferation of rumen bacteria, while the growth of rumen fungi was inhibited in this result, which may be due to the antagonism between rumen bacteria and fungi. The growth of cellulose-degrading fungi was inhibited to some extend after the addition of MOS in the diet. Candida and Zopfiella, closely related to mycotic mastitis and immunosuppression respectively, had decreasing trends in rumen with supplemental MOS in the diets of the dairy cows, indicating that MOS might play a positive role in inhibiting harmful fungi. The experimental result shows that mannan oligosaccharides in the diet of dairy cows inhibited the growth of cellulolytic fungi and the proliferation of harmful fungi. In addition, the results showed that there might be the antagonistic effect between rumen fungi and bacteria potentially, which should be focused on for further study