Reconstituted tobacco sheet process has been developed to treat and reuse tobacco wastes in the industry. During this process, microorganisms in original and concentrated tobacco waste extract (TWE) might play important roles in the final quality of the reconstituted tobacco. However, microbial communities in TWE remain largely unknown. In the present study, the Roche 454 bar-coded pyrosequencing was applied to analyze the bacterial community structure in samples. Comparison based on 16S rRNA gene sequences showed that the original and concentrated solutions of TWE harbored abundant bacteria probably resistant to the acid, high nicotine concentration, and high osmotic pressure environment. The dominant phyla were Firmicutes and Proteobacteria. Lactobacillus and Lysinibacillus were the dominant genera of Firmicutes. The most interesting genus of Proteobacteria was Pseudomonas. It is the first time to reveal the bacterial diversities on the TWE samples from the process of reconstituted tobacco sheets.