Root rot is one of the most serious diseases in the cultivation of Atractylodes lancea (Thunb.) DC.. Arbuscular mycorrhizal fungi (AMF) can coexist with more than 90% vascular plants and have a certain inhibitory effect on plant soil borne diseases. A pot experiment was conducted to investigate the effects of Glomus mosseae (Gm), Glomus intraradices (Gi) and Glomus etunicatum (Ge) on the seedling growth and the bio-effects of AMF against root rot of A. lancea (Thunb.) DC.. The results showed that the mycorrhizal infection rates of A. lancea (Thunb.) DC. seedlings in the three inoculation groups were in the order of Gm (60.44%), Ge (55.89%) and Gi (52.33%). All of the three AMFs could promote the vegetative growth of A. lancea (Thunb.) DC. seedlings, with Ge group showing the best performance. G. mosseae had a greater promoting effect on the underground material accumulation of A. lancea (Thunb.) DC. seedlings than Ge or Gi, with the mycorrhizal dependency of fresh weight and dry weight of underground part to mycorrhiza was 260.34% and 285.45%, respectively. In addition, the three AMFs could reduce the disease index, and improve the activities of the root disease resistance enzymes PAL, β-1,3-glucanase and chitinase. Namely, AMF could reduce the occurrence of root rot of A. lancea (Thunb.) DC. seedlings, among which G. mosseae and G. etunicatum had better control effects, with 53.15% and 62.94%, respectively. The results indicated that AMF can promote the vegetative growth of A. lancea (Thunb.) DC. seedlings and reduce the occurrence of root rot, and G. mosseae and G. etunicatum can be used as alternative biological agents for the control of root rot of A. lancea (Thunb.) DC..