Chromolaena odorata (L.) King & H. Rob. is a perennial ubiquitary aromatic herb common in South East Asia, West Africa and parts of Australia. Recently, C. odorata leaf extract has been demonstrated to exhibit antibiofilm activity against Pseudomonas aeruginosa. However, the molecular mechanism underlying this biological activity has not been elucidated. To address this issue, the cytoplasmic protein expression of P. aeruginosa biofilm following treatment with C. odorata chloroform extract (COCE) was investigated. Based on SDS polyacrylamide gel electrophoresis, the treatment of COCE resulted in changes in the profile of cytoplasmic proteins of P. aeruginosa biofilm under aerobic and anaerobic conditions. Densitometric analysis between 400 and 750 nm successfully revealed a number of differentially expressed cytoplasmic proteins (under aerobic condition: 23 kDa, 35 kDa and 46 kDa; under anaerobic condition: 45 kDa) in P. aeruginosa biofilm which indicated the antibiofilm effects of COCE. We suggest that the metabolically important cytoplasmic proteins in P. aeruginosa biofilm may be involved in the antibiofilm mode of action of COCE. Nevertheless, the identity of these cytoplasmic proteins remains to be further investigated