Abstract: In order to enrich the resources of highly efficient antagonistic microorganisms against Thielaviopsis basicola, highly efficient antagonistic strains were isolated from the inter-root soil of Yuxi tobacco planting areas, and were identified by combining physiological and biochemical characteristics and 16S rDNA sequence analysis, and the potential of antagonistic effects against Thielaviopsis basicola in vitro was assessed through the study of antagonistic activities in the fermentation broth of the bacterial strains. The results demonstrated that among the screened 12 antagonistic strains, YG-1 exhibited the strongest antagonistic activity. This strain was identified as Pseudomonas fluorescens based on its physiological, biochemical characteristics, and 16S rDNA sequencing results. Fermentation broth produced by strain YG-1 effectively inhibited the growth of Thielaviopsis basicola, leading to distorted and abnormal hyphal growth, as well as reduced sporulation. Acids, aldehydes, esters, and several sulfur-containing compounds were determined by GC-TOF-MS, and the effective fermentation broth inhibitory substances were revealed to be primarily acid compounds and sulfur-containing compounds. The fermentation filtrates of strain YG-1 contained inhibitory substances that disrupted the spore membrane of Thielaviopsis basicola. At a concentration of 0.25 mg/mL, the spore membrane rupture rate reached 55.42%, accompanied by noticeable leakage of ions and nucleic acids. The antagonistic potential of strain YG-1 highlights its value for the development of biocontrol agents against Thielaviopsis basicola.