A rare plant species of the Orchidaceae family, Dendrobium officinale is considered among the top ten Chinese medicinal herbs for its polysaccharide. Since 2021, when the dieback disease of D. officinale was first reported in Yueqing City, Zhejiang Province, China, Fusarium isolates (number = 152) were obtained from 70 plants in commercial greenhouses. The disease incidence ranged from 40% to 60% in the surveyed areas. Multilocus sequence analysis (MLSA) coupled with morphological characterization revealed that the collected isolates belonged to five species (sp.), viz., Fusarium concentricum, F. fujikuroi, F. nirenbergiae, F. curvatum, and F. stilboides, with isolation frequencies of 34.6%, 22.3%, 18.4%, 13.8%, and 10.5%, respectively. Notably, at least two Fusarium species were simultaneously isolated and identified from the infected plants. Finally, the pathogenicity test results demonstrated that such species were responsible for the dieback disease of D. officinale. However, F. concentricum and F. fujikuroi were more invasive compared to the other species in this study. To the best of the authors’ knowledge, this study was the first report of F. concentricum, F. curvatum, F. fujikuroi, F. nirenbergiae, and F. stilboides causing the dieback disease of D. officinale in China and worldwide. This work provides valuable data about the diversity and pathogenicity of Fusarium populations, which will help in formulating effective strategies and policies for better control of the dieback disease.