Cucumber is one of the top ten vegetables globally and is widely cultivated worldwide. However, Fusarium wilt, caused by Fusarium oxysporum f. sp. Cucumerinum, is one of the most serious soil-borne diseases in cucumber cultivation, causing significant economic losses. Biological control has great potential in the prevention of cucumber wilt disease, but the mechanism involved still needs further research. In this study, biocontrol isolate Bacillus subtilis 1JN2, which was isolated in our previous work, was evaluated in field conditions against Fusarium wilt, and the rhizosphere fungal diversity was analyzed. The results indicated that the biocontrol efficacy of B. subtilis 1JN2 reached 58.5% compared with the blank control, and the population density of F. oxysporum in the rhizosphere decreased from 495 copies/g of soil before inoculation to 20 copies/g 14 days after treatment. High-throughput sequencing demonstrated that after an inoculation of 1JN2, the populations that decreased significantly include the genera of Olpidium and Pseudallescheria, from more than 20% to less than 8%. And the most increased population belonged to the family Chaetomiaceae, from 6.82% to 18.77%, 12.39%, 44.41%, and 19.41% at the four sample time points after treatment. In addition, soil-related enzyme activities, including catalase, soil dehydrogenase, alkaline phosphatase, and polyphenol oxidase, were analyzed before and after treatment with 1JN2. The results indicated that all the enzyme activities showed an upward trend following inoculation. These findings demonstrate the potential of using B. subtilis 1JN2 as a biocontrol agent for controlling Fusarium wilt in cucumber.