Bioaugmentation can alter the potential activity as well as the composition of the naturally occurring microbial biota during bioremediation of a contaminated site. The focus of the current study is the pollutant 17β-estradiol (E2), which can cause endocrine effects and is potentially harmful to aquatic biota and to public health. The community composition and function of biofilms, originating from a wetland system, as affected by augmentation of an estradiol-degrading bacterium (EDB-LI1) under different conditions, were investigated. EDB-LI1 inoculation into biofilm from two wetland ponds representing early and advanced water treatment stages, respectively, yielded three significant observations, as follows: (i) EDB-LI1, enriched from a biofilm of a constructed wetland wastewater treatment system, was detected (by quantitative PCR [qPCR] analysis) in this environment in the augmented biofilm only; (ii) the augmented biofilm acquired the ability to remove estradiol; and (iii) the bacterial community composition (analyzed by PCR-denaturing gradient gel electrophoresis [DGGE]) of the augmented biofilm differed from that of the control biofilm. Furthermore, EDB-LI1 bioaugmentation showed a higher level of removal of estradiol with biofilms that originated from the advanced-treatment-stage wetland pond than those from the early-treatment-stage pond. Hence, the bioaugmentation efficiency of EDB-LI1 depends on both the quality of the feed water and the microbial community composition in the pond.