This study explored the potential to improve the storage quality and prolong the shelf life of truffles by storing them in a modified atmosphere fresh-keeping box with sealed gas components of Active Modified Atmosphere Packaging (AMAP, 40% O2 + 60% CO2) at 4 °C. During the storage period, a total of 63 volatile components in 10 categories were detected, with aldehydes being the most abundant and the relative content of ethers being the highest. The relative odor activity value and principal component analysis revealed that isovaleraldehyde, 1-octen-3-ol, 1-octen-3-one, and dimethyl sulfide were the characteristic flavor components of fresh truffles. However, 3-methylthiopropionaldehyde and (E, E)-2,4-nonadienal were the components that caused the deterioration of truffle flavor and could potentially serve as markers of truffle decay characteristics. 16S rDNA high-throughput sequencing showed that Leuconostoc and Lactococcus were dominant in the truffle samples stored for 14 days, but the abundance of putrefactive pathogenic bacteria showed an increasing trend in the truffle samples stored for 28 days. During the whole storage period, the common fungi detected in the different treatment groups were Candida and Aspergillus. The relative abundance of the former decreased, while the relative abundance of the latter decreased initially and then increased. The correlation between volatile components and the microbial flora was further analyzed, which indicated that Lactococcus and Lactobacillus had the same contributions to the same flavor, while Pseudomonas and Glutamicibacter had the opposite contributions to the same flavor. The results provide a reference for the storage and preservation of truffles.