Abstract: To clarify microbial community succession and the inhibitory effect of potassium sorbate on the dominant spoilage microbes of the tobacco casing during storage, metagenomic and molecular biological technologies were employed to systematically investigate the dynamic changes in microbial community, identify the dominant spoilage microorganisms, elucidate its biological characteristics, and further evaluate the efficacy of potassium sorbate against the strain. The results showed that on the 7th day of storage, the total viable count and yeast count in the tobacco casing increased by 4.7×103 CFUmL–1 and 2.8×103 CFUmL–1, respectively, compared to the 5th day. This was accompanied by deterioration phenomena such as a sour odor, gas production, and the formation of a yellow microbial film. During 0-5 days of storage, microbial species richness and diversity of the tobacco casing showed an initial upward trend but significantly decreased by day 7, with reduced community evenness. The microbial community structure shifted to be dominated by a limited number of species, notably the genus Wickerhamomyces. The dominant spoilage microorganism was identified as Wickerhamomyces anomalus, with optimal growth at 30 °C and pH 6.0. Sucrose, fructose, and glucose were identified as its preferred carbon sources. The antibacterial results revealed that the inhibitory effect of potassium sorbate on W. anomalus exhibited concentration-dependence. At a concentration of 1.00 mg·mL–1, a significant delay in the growth of the strain was observed. As the concentration of potassium sorbate increased in a gradient manner, the growth of the strain gradually stagnated, and the minimum inhibitory concentration (MIC) was determined to be 8.00 mg·mL–1.