BACKGROUND The antimicrobial mechanisms of ε-polylysine (EPL) against Pseudomonas aeruginosa and Aspergillus fumigatus biofilm were investigated. MATERIAL AND METHODS We assessed the changes in electric conductivity of broth and total sugar concentration, as well as changes in phosphorous metabolism and protein expression, of the 2 organisms before and after treatment with EPL. RESULTS The experimental results showed that EPL has antimicrobial activity against Pseudomonas aeruginosa and Aspergillus fumigatus, but the activity was much stronger for the former. After treatment with EPL, the electric conductivity and total sugar concentration of microbial broth increased, suggesting that EPL damages the cell membrane structure, which increases permeability of the cell membrane and release of cell components. CONCLUSIONS The consumption of phosphorous decreased in the EPL-treated organisms, which seriously affected the synthesis of important cell components such as nucleic acid and phospholipid, as well as energy metabolism.