[Background] Tobacco flavor is an important auxiliary material in cigarette production. Microbial contamination usually occurs in the processes of transportation and storage of tobacco flavor, especially the residual casing, which could cause spoilage of tobacco flavor. [Objective] To investigate the diversity of spoilage microorganisms in the deteriorated tobacco flavor and identify the main spoilage microorganisms causing the deterioration of tobacco flavor. [Methods] Dilution plate counting method was applied to monitor the microbial composition of the deteriorated tobacco flavor from Yunnan, Fujian and Guangxi of typical hot-humid areas in China, and then the 16S rRNA, ITS or 26S rRNA genes of the isolates were sequenced. Furthermore, verifiable experiments were used to identify the main spoilage microorganisms causing deterioration of the tobacco flavor. [Results] The tobacco flavor samples from the three typical hot-humid areas in China were with different degree of bacteria, molds or yeasts contamination, and the bacterial contamination was the most common. A total of 76 bacterial strains, 9 fungal strains and 42 yeast strains were isolated. The 76 bacterial strains were distributed in 2 phyla, 4 classes, 5 orders, 10 families and 15 genera. Ralstonia, Citrobacter, Enterobacter and Bacillus were the dominant genera. The 9 fungal strains were distributed in 3 classes and 6 genera of the Ascomycota phyla, with 1?2 fungal strains belonging to each genus. The 42 yeast strains were distributed in 6 genera of Ascomycota. Pichia, Wickerhamomyces and Saccharomyces were the dominant genera. The bacterial composition of Yunnan and Fujian samples was obviously more than that in Guangxi. The fungal composition of Guangxi and yeasts’ composition of Fujian were more than those in the other two sites, respectively. The verifiable experiment results showed Citrobacter, Enterobacter, Bacillus, Ralstonia and Pichia were the main spoilage microorganisms in the tobacco flavor samples. [Conclusion] The study on the composition of contaminated microorganisms and the confirmation of spoilage microorganisms in the deteriorated tobacco flavors could help to control the contamination of microorganisms and the spoilage of tobacco flavors in hot-humid areas in China.