Abstract: To identify the causal pathogens of alfalfa root rot with strong pathogenicity, pathogens were isolated from alfalfa (Medicago sativa) plants with alfalfa root rot growing in Wuwei City, Gansu Province. The pathogens were identified based on their morphology and by molecular methods. The results showed that the strain MXGF7 caused acute root rot of alfalfa. Most of its macroconidia were fusiform or sickle-shaped (3.3-4.7 μm×14.8-32.0 μm), and its microconidia were fusiform or dumbbell-shaped (2.9-4.2 μm×8.4-18.2 μm). The ITS sequence and the Fu3/Fu4 specificity gene sequence of the pathogenic fungus shared 99% similarity with those of Fusarium chlamydosporum, and so the pathogenic fungus was identified as F. chlamydosporum. We isolated 123 endophytic bacterial strains from alpine grassland in the Eastern Qilian Mountains, and evaluated their ability to antagonize the pathogenic strain of F. chlamydosporum. Sixty-one of the bacterial strains showed an antifungal ratio greater than 55%. The most antagonistic bacterium, strain 265ZY3, was identified as Bacillus amyloliquefaciens based in its morphology and 16S rDNA gene sequence.