Abstract: Peanut southern blight, a soil-borne fungal disease caused by the infection of Sclerotium rolfsii Sacc., has become an important threat to peanut production. In order to explore the antagonistic bacteria with good control effect on peanut southern blight, 570 strains of bacteria were isolated from the rhizosphere of S. rolfsii infected plants by dilution coating method, and 32 strains of S. rolfsii antagonistic bacteria were screened by plate face-off method. 16S rRNA gene sequence, morphology, physiological and biochemical characteristics were combined to determine the taxonomic status of bacteria. Antagonistic bacteria was co-cultured with pathogen to determine the inhibitory effect of antagonistic bacteria against pathogen. The biocontrol and growth promotion effect of antagonistic bacteria were confirmed by pot experiment. The results showed that among the 32 antagonistic strains, 4 strains HNZZ23, HNKF5, HNXX14 and HNXX169 has a long inhibitory time and good inhibitory effects on S. rolfsii, and the inhibition rates of the fermentation broth on the mycelial growth of the pathogenic strain were 85.30%, 85.88%, 85.16% and 91.00%, respectively. The maximum inhibition rates of sclerotium germination were 100%, 85.33%, 82.67% and 93.33%, respectively. The results of pot experiment showed that the control effects of strain HNZZ23 and HNXX14 on peanut southern blight were 30.50% and 25.73%, respectively, and strain HNZZ23 significantly promoted the growth of peanut plants. After taxonomic identification, strains HNZZ23, HNKF5, HNXX14 and HNXX169 were identified as Bacillus atrophaeuse, B. pumilus, Pseudomonas aeruginosa, and B. subtilis, respectively. The results of this study showed that the rhizosphere strains HNZZ23, HNKF5, HNXX14 and HNXX169 isolated from the infected peanut has good antagonistic effects and biocontrol potential against S. rolfsii, which provide high quality strain resources for the biological control of peanut southern blight disease.