D-amino acids can inhibit the formation of biofilm without affecting bacterial growth, which is a new strategy to control membrane biofouling. This paper investigated the effects of D-tyrosine on the performance of membrane bioreactor (MBR) during short-term operation. The results showed that the critical flux of activated sludge in MBR increased with the existence of D-tyrosine. The removal efficiencies of wastewater were not affected by D-tyrosine. The fouling propensities of soluble microbial products (SMP) in both MBRs were almost the same. Extracellular polymeric substance (EPS) in D-tyrosine group exhibited weaker fouling propensity. Further research indicated that the addition of D-tyrosine did not have a significant impact on SMP. D-tyrosine lead to the reduction of EPS polysaccharides and the great fluctuation of EPS protein. Microbial community analysis demonstrated that the addition of D-tyrosine changed the microbial community structure in MBR, which might be one of the reasons for the change of EPS.