The membrane bioreactor (MBR) has recently been the focus of research for the treatment of emerging contaminants such as antibiotics in wastewater. Although the biofilm on membrane in an MBR has been considered a cause of “membrane biofouling”, its positive impact on removing pollutants has not been well-studied. This study was designed to investigate the retention effect on the permeation of ampicillin (AMP) by the biofilm coated on cellulose acetate (CA) membrane (commonly used for MBRs) utilizing a novel method based on microbial sensitivity test. The bioflim layer (thickness of 12–16μm) increased the resistance of the membrane for AMP permeation by 3–28%. Diffusion appeared to be the main driving force for the mass transfer of AMP across the membrane. Besides, the biofilm increased the retention of AMP by 23% but exhibited similar adsorption capacity with comparison of the suspended activated sludge, which indicates that the compact structure of the biofilm was the major contributor for the added retention effect on AMP by the biofilm-coated CA membrane. This study suggests that biofilm (biofouling) in MBRs increases the retention of small-molecule constituents such as antibiotics. A delicate tradeoff between reduced wastewater throughput and increased retention of contaminants should be obtained when an MBR is designed and operated.