Abstract: Tobacco black shank, caused by Phytophthora parasitica var. nicotianae infection, is a major soil-borne disease of tobacco. To achieve the sustainable and effective management of this disease, this study screened biocontrol strains from different tobacco-growing regions, evaluated their stress tolerance, identified their taxonomic status, determined their antifungal mechanism against the target pathogen, and assessed their disease-suppressive and plant growth-promoting activities. The results revealed that a total of four biocontrol strains with high antifungal activity were isolated and screened. Among these strains, DY-6-6 exhibited the optimal antifungal activity and stress tolerance, and was identified as Bacillus velezensis. Treatment with B. velezensis DY-6-6 led to an increase in malondialdehyde (MDA) content, a decrease in superoxide dismutase (SOD) activity, catalase (CAT) activity, and protein content of P. parasitica, as well as a high biofilm-forming capacity. B. velezensis DY-6-6 exerted both germination-promoting and growth-promoting effects on tobacco, as the seed germination rate, leaf area, plant height, and biomass were significantly increased after treatment. The relative preventive effect of B. velezensis DY-6-6 fermentation broth on tobacco black shank in pot experiments under controlled indoor conditions was 70.35%, and the relative therapeutic effect was 59.20%, which was consistent with that of the fungicide. These results provide a new biocontrol strain for the management of tobacco black shank and clarify its disease-suppressive and growth-promoting mechanisms.