Two lab-scale nitrifying sequencing batch biofilm reactors, with (SBBR_CN) or without the addition of organics (SBBR_N), were operated to investigate potential roles of acyl homoserine lactone (AHL) based quorum sensing. AHLs of N-[(RS)-3-Hydroxybutyryl]-L-homoserine lactone, N-hexanoyl-L-homoserine lactone (C-6-HSL) and N-octanoyl-L-homoserine lactone (C-8-HSL) were detected in both reactors. C-6-HSL and C-8-HSL were also detected in batch experiments, especially with stimulated nitrite oxidizing bacteria activities. Quorum sensing affected biofilm formation mainly through the regulation of extracellular protein production. By the metagenomics analysis, many identified genera and species could participate in quorum sensing, quorum quenching and extracellular polymeric substances (EPS) production. A high quorum quenching activity was obtained in SBBR_CN, whereas a high quorum sensing activity in SBBR_N. Nitrosomonas-like ammonia oxidizing bacteria, Nitrospira-like nitrite oxidizing bacteria and Comammox harbored genes for AHL synthesis and EPS production. Possible relationships among AHLs synthesis, biofilm formation and nitrifiers activity were proposed.