Abstract: Black rot, caused by Fusarium oxysporum, is a significant disease affecting the production and cultivation of Echeveria succulents. To manage this disease, we isolated and identified an endophytic Bacillus subtilis strain, OLR3-9, from the roots of wild rice (Oryza longistaminata). The strain's biocontrol efficacy was evaluated through in vitro antifungal activity, greenhouse trials, and colonization assessments. Results demonstrated that OLR3-9 effectively controls black rot in two Echeveria varieties, ‘Romeo Rubin’ (E. agavoides) and ‘Jing Ye’ (E. derenbergii), with mycelial inhibition rates of 63.45% and 66.60%, spore inhibition rates of 78.74% and 76.45%, and pot trial control effects of 65.55% and 73.13%, respectively. Interaction with OLR3-9 resulted in mycelial deformation and reduced sporulation of the pathogen, alongside stable colonization of the host plant's roots, stems, and leaves, thereby preventing pathogen invasion and spread. The Bacillus subtilis strain OLR3-9 shows strong potential as a biocontrol agent against black rot in Echeveria succulents.