Abstract: To achieve the green prevention and control of tobacco black shank, Phytophthora nicotianae was isolated using tissue analysis combined with molecular identification. Through pathogenicity tests and oomycete virus detection, a hypovirulent strain XC-26-5 carrying a virus was screened. Following shake-flask cultivation, the microbial agent was prepared via emulsification and shearing. Field experiments were conducted to investigate the trends and differences in the effects of different application rates of the biocontrol agent on the abundance of P. nicotianae in soil, incidence rate, disease index, and control efficacy. Amplicon sequencing was employed to analyze its impact on the diversity and community structure of tobacco rhizosphere soil microorganisms. Results showed that the hypovirulent strain XC-26-5 grew slowly on PDA medium, with shorter sporangiophores and reduced sporangia, and carried the partitivirus PnPV1 (Phytophthora nicotiana partitivirus 1). Field trials indicated that when the XC-26-5 agent was applied at 4.50 kg/ha, the soil pathogen population decreased by 50.1%, the incidence of black shank was reduced by 69.73%, and the control efficacy reached 70.1%. High-throughput sequencing revealed that XC-26-5 significantly increased the Simpson, Chao1, and ACE indices of soil microbial diversity, altered microbial community structure, and enriched beneficial genera such as Sphingomonas and Lysobacter in the rhizosphere. The virus PnPV1 carried by XC-26-5 effectively reduced the pathogenicity and sporulation of P. nicotianae, achieving robust control of tobacco black shank disease. Application of XC-26-5 at 4.50 kg/ha demonstrated optimal efficacy in pathogen suppression and soil microbiome modulation.