Aims This study was designed to evaluate the biocontrol of the arbuscular mycorrhizal fungus (AMF) Funnelliformis mosseae and the rhizobium Sinorhizobium medicae on alfalfa (Medicago sativa) wilt caused by Fusarium oxysporum, a severe soil-borne fungal pathogen. Methods and Results The effects of co-inoculation of F. mosseae and S. medicae on alfalfa growth, nitrogen, phosphorus uptake and wilt caused by F. oxysporum were tested. Plant defence-related chemicals were measured to reveal the biochemical mechanism by which alfalfa responds to pathogen infection and how it is regulated by AMF and rhizobium. Pathogen infection caused typical yellowing of alfalfa leaflets and significantly reduced plant AMF colonization. AMF or rhizobium alone and the co-inoculation reduced the plant disease index by 83 center dot 2, 48 center dot 4 and 81 center dot 8% respectively. Inoculation with AMF or rhizobium alone increased the dry weight of alfalfa by more than 13 and 3 times respectively; it also increased plant chlorophyll content by 65 center dot 6 and 16 center dot 6% respectively. Co-inoculation of AMF and rhizobium induced the plant to accumulate more disease-related antioxidant enzymes, plant hydrolase and plant hormones, such as superoxide dismutase, beta-1,3-glucanase, chitinase, and phenylalanine ammonialyase, abscisic acid, ethylene and H2O2, under pathogen stress. Conclusions Co-inoculation with F. mosseae and S. medicae offered complementarily improved alfalfa nutrient uptake and growth, which increased plant health. The co-inoculation of AMF and rhizobium regulated plant physiological and biochemical processes and induced plants to produce defence-related compounds, thus decreasing the severity of disease. The simultaneous application of F. mosseae and S. medicae is a potential biocontrol strategy to increase the systemic defence responses of alfalfa to Fusarium wilt. Significance and Impact of the Study This research showed that complex plant-pathogen interactions are affected by rhizobium and AMF, providing insight into plant-microbiome interactions in the rhizosphere as well as the application of the microbiome in agriculture production.