Fusarium wilt disease, caused by Fusarium oxysporum f. sp. cucumerinum (FOC), leads to widespread yield losses and quality deterioration in cucumber. Endophytes, as environmentally friendly control agents that enhance pathogen resistance in their host plants, may mitigate these problems. In this study, we isolated 14 endophytic bacteria from invasive Ambrosia artemisiifolia and screened the strain Bacillus subtilis TCX1, which exhibited significant antagonistic activity against FOC (inhibitory rate of 86.0%). TCX1 killed Fusarium oxysporum by being highly likely to produce lipopeptide and producing wall hydrolytic enzymes including protease, cellulase, and β-glucanase, thereby inhibiting mycelial growth and spore germination and causing peroxidation of FOC’s cytoplasmic membrane. In addition to its direct effects, TCX1 exerts indirect effects by inducing cucumber resistance to FOC. When cucumber seedlings were inoculated with TCX1, antioxidant enzymes related to disease resistance, including Superoxide dismutase (SOD), Peroxidase (POD), Polyphenol oxidase (PPO) and Phenylalanine ammonialyase (PAL) in cucumber, were significantly increased. The marker genes involved in induced systemic resistance and the salicylic acid signaling pathway, such as npr1, pr1a, pr2, pr9, lox1, and ctr1, were also dramatically upregulated, indicating these pathways played an important role in improving cucumber resistance. Notably, TCX1 can also promote cucumber growth through producing indole-3-acetic acid, solubilizing phosphate, and secreting siderophores. Given that TCX1 has dual functions as both a biological control agent and a biofertilizer, it offers an effective strategy for managing cucumber seedling blight while enhancing plant productivity.