The antimicrobial mechanisms of tea polyphenol against Gram-positive S. aureus and Gram-negative P. aeruginosa were investigated by determining the changes in electric conductivity and total sugar concentration of broth for bacteria treated by tea polyphenol, as well as the changes in phosphorous metabolism and protein expression of bacteria. As the results showed, tea polyphenol had antimicrobial activity against S. aureus and P. aeruginosa, but stronger against the former. The facts that the electric conductivity and total sugar concentration of microbial broth increased indicated that tea polyphenol could damage the structure of cell membrane, which resulted in the increase of permeability of cell membrane and release of cell components. Besides, the consumption of phosphorous decreased in the tea-polyphenol-treated bacteria, which seriously influenced the synthesis of important cell components such as nucleic acid and phospholipid and energy metabolism. SDS-PAGE assay demonstrated that tea polyphenol could block the protein expression in bacteria, which influenced the cell structure composition and catalyzing activity of enzyme, and finally leading to the lost of normal physiological function of bacterium.