In this study, both traditional colony counting and PCRDGGE (PCRDenaturing gradient gel electrophoresis) technology were used to analyze the quantity and species characteristics of microorganisms in the microenvironment, Chongqing China Three Gorges Museum. The results showed that the species and quantity of cultivable microorganism in the air of the museum microenvironment was significantly higher in summer than in winter. Species of Acinetobacter, Staphylococcus、Pseudomonas and Advenella were found to predominate. In addition, Acinetobacter sp. and Micrococcus sp. were dominant in summer while Bacillus sp. predominated in winter. The results were helpful in terms of evaluating potential microbial pollution and to providing scientific evidence for preventive protection of exhibited or collected cultural relics in museums.