The diseased soil containing pathogens of Fusarium oxysporum f. sp. was used as the material. Using the pot experiment to explore the control effects of Bacillus megaterium B213, Bacillus polymyxa B207, and Bacillus subtilis B204 alone or in combination on cucumber fusarium wilt and its mechanism. The results showed that the three microbial agents alone or in combination could effectively inhibit the occurrence of cucumber fusarium wilt and significantly increase the biomass of cucumber. The effect of the two microbial agents in combination was better than that of single microbial agent. And the effect of the three microbial agents in combination was better than that of the two microbial agents. 35 days after transplanting cucumber seedlings, compared with the control group, the incidence of cucumber fusarium wilt in the three-agent combination group was reduced by 79.99%; the dry weight of cucumber plants was increased by 6.92 times; the activities of plant resistance enzymes, such as acid lyase, polyphenol oxidase, and peroxidase increased by 60.02%, 28.90%, and 108.60% respectively; the number of rhizosphere soil Fusarium oxysporum decreased by 2 orders of magnitude. These effects may be related to the antagonism of these three Bacillus agents to pathogenic bacteria, phosphorus dissolving ability, and secretory ability of indoleacetic acid, protease, chitinase and ferrophilin.