In this study, the impact of dead cells on biofouling and consequent biofouling effects on three pharmaceutically active compounds (PhACs) retention by nanofiltration (NF) and reverse osmosis (RO) membrane were systematically studied through inoculating live and dead Pseudomonas aeruginosa into artificial wastewater, respectively. The biofilm was characterized by quantifying the surface properties, extracellular polymeric substances (EPS) and total organic carbon (TOC) amount, biomass activity, total live/dead cells and biofilm thickness. The solution-diffusion model was applied to identify the role of biofilm on the solute retention. The results showed that dead cells in feed water had the same potential to cause biofouling as live cells. After excluding the flux effect, we observed an increase in PhAC rejection during biofouling with live cells due to the dominant steric exclusion. However, PhAC rejection decreased significantly during biofouling with dead cells. It was explained that dead cells could enhance cell activity in feed water and increase total biomass, cell counts and biofilm thickness on membrane surfaces, which improved the biofilm enhanced osmotic pressure (BEOP). Our study will give implication in further research on the dead cells in feed water and the removal of PhACs by NF/RO membrane under the biofouling condition.