Display omitted •EPS, microbial communities and its implications for clogging were investigated.•Total EPS quantity increased in SSFCWs by applying aeration with biochar addition.•Contents of different types of EPS decreased from upper to lower substrate layer.•Biochar and aeration enhanced the abundance of microbes but reduced the diversity.•Aeration and biochar addition may increase potential risks of clogging in SSFCWs. Substrate clogging is regarded as one of the main influencing factors on the sustainable operation in subsurface flow constructed wetlands (SSFCWs). The accumulation of extracellular polymeric substances (EPS) produced by microbial metabolism may contribute to the substrate clogging due to the increasing formation of biofilms in CWs. In this study, the quantity and composition of EPS, and the distribution and variation of bacterial communities in different CW systems with and without aeration and biochar addition were comparatively investigated. Biochar and intermittent aeration addition could shift the production and compositions of EPS in SSFCW. Higher total EPS production was observed in CW with intermittent aeration and biochar (328 mg L−1), compared with control (241 mg L−1), CW with biochar addition (154 mg L−1) and CW with intermittent aeration (285 mg L−1). In addition, biochar addition could decrease the content of soluble EPS (S-EPS), and combination of biochar and aeration could increase the content of tightly B-EPS (TB-EPS). Four fluorescent components were identified in EPS samples by parallel factor (PARAFAC) analysis. The fluorescence intensity of tryptophan-like substance was maximum, followed by tyrosine-like substance and humic acid-like substance. The application of intermittent aeration and biochar improved the abundance of microbes but decreased the diversity of microbes. Moreover, higher relative abundance of some functional bacteria correlated with the nitrogen removal was found in biochar added intermittently aerated SSFCW. The obtained results revealed that intermittent aeration and biochar addition could enhance treatment performance, but might have a potential risk of substrate clogging in SSFCWs.