Abstract: In order to improve the biomass of Trichoderma asperellum HG1 and Bacillus subtilis tpb55 in the co-culture system, and increase their anti-oomycetes activities against Phytophthora nicotianae, and to explore the bioactive ingredients, the solid-state fermentation system was constructed. The anti-oomycetes activities were evaluated by the mycelium growth rate method. The solid-state fermentation conditions were determined by the anti-oomycetes activities and biomass of HG1 and tpb55. The bioactivity guided separation and isolation of fermentation extracts by silica gel and octadecylsilyl silica gel (ODS) column chromatography (CC), as well as high performance liquid chromatography (HPLC), together with NMR and ESI mass spectrometry were applied to clarify and identify the bioactive ingredients. Finally, pot experiments were applied to verify the control effect of solid-state co-culture of HG1 and tpb55 on tobacco black shank. The results showed that the optimal solid-state fermentation condition was as follows:the inoculation sequence was first B. subtilis (10⁶ CFU/mL, 3%) 12 h and then inoculated with T. asperellum (10⁶ CFU/mL, 1.5%). After 14 d of fermentation, under the optimal condition, the bacterial amounts of tpb55 were 1.58×10¹⁰ CFU/g and the spore amounts of HG1 were 6.75×10¹⁰ CFU/g, with the strongest anti-oomycete activity towards P. nicotianae (inhibition rate 73.25%). A pure compound was isolated from the bioactive fraction and identified as harziandione. The results of pot experiments showed that the control effect of solid co-culture strains on tobacco black shank was significantly better than that of single culture, reached 61.94%. The co-cultured strains could also improve soil physical and chemical properties. This study provided a new idea and method for the research and development of co-culture biocontrol agents for tobacco black shank disease.