Abstract: In order to reveal the surface microbial changes of Tuber indicum during storage at 4 ℃, identify the dominant spoilage bacteria and select the natural antibacterial agent with good antibacterial effect. In this paper, high-throughput sequencing and traditional culture technology were used to study the surface flora structure and dominant spoilage bacteria of T. indicum during storage. Five natural plant essential oils were selected to study their inhibitory effects on dominant spoilage bacteria by measuring inhibitory rate and calculating their EC₅₀ value. The results showed that the total number of colonies on the T. indicum surface was gradually increasing during storage, and reached 2.28×10⁶ CFU/g on the 15th day which appeared odorous, sticky or moldy phenomena. Then reached the end of shelf life on the 30th day. Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes and Verrucomicrobia were the dominant phyla during storage. Pseudomonas、Rhizobium、Pedobacter、Sphingobacterium、Flavobacterium、Serratia、Janthinobacterium、Sphingopyxis and Variovorax were the dominant genera. Two strains of bacteria were isolated which could cause truffle decay rate of more than 90%, and identified as Bacillus subtilis and Brachybacterium faecium. Among the 5 essential oils, lemon grass essential oil had the best antibacterial effect on B. subtilis and the EC₅₀ value was 5.471 μL/L, thyme essential oil had the best antibacterial effect on B. faecium and the EC₅₀ value was 6.350 μL/L. It provides a theoretical basis for the further compound application of lemon grass and thyme essential oil in the postharvest storage and prolong shelf life of truffles.