Chemical fungicides are a frequently used intervention for the control of the Fusarium wilt of chrysanthemum, but are no longer considered environmentally friendly. However, the biofungicides offer one of the best alternatives to reduce the use of chemical fungicides. In this study, a series of two-year greenhouse experiments were conducted to evaluate the soil-applied chemical fungicide (dazomet, DZ) and biofungicide (biocontrol agent combined with B. subtilis NCD-2, BF) for controlling the Fusarium wilt of chrysanthemum and its effects on rhizosphere soil microbiota. The results indicated that DZ and BF showed good control efficacy of Fusarium wilt of chrysanthemum in the two-year application evaluation. However, the DZ treatment significantly decreased the soil catalase and urease activities compared with the control, while BF showed a significant increase in bacterium/fungus ratios (B/F), soil urease and acid phosphatase activities. Abundances of potential plant pathogens F. oxysporum, Rhizoctonia zeae and Rhizoctonia solani were also lower, while potential plant-growth-promoting micro-organisms like the Rhizobiales bacterium and Mariniflexile sp. were higher in the BF treatment than in the control. Our findings suggested that the overall effect of the soil biota on chrysanthemum growth was more positive and stronger in the BF treatment than in the DZ treatment.