The flow cell system and confocal laser scanning microscopy were applied to investigate the characteristics of biofilm formed by the nitrogenous heterocyclic compound (NHC)-degrading bacteria under different environmental conditions. The results showed that increasing the initial inoculum density and prolonging the initial attachment time were beneficial to the adhesion and biofilm formation of the NHC-degrading bacteria on the surface of the substrate. When the flow rate of the medium was reduced, the biofilm became more homogeneous and formed more water channel structures. Moreover, the biofilm under different conditions presented a same phenomenon that the inner layer (near the surface) had a low ratio of living cells and the outer layer (distant from the surface) had a high ratio of living cells. Compared with the single-strain biofilm, the dual-strain biofilm had an advantage in thickness, surface coverage ratio and living cell ratio. The target NHC concentration also had a significant effect on the morphology and cell viability of the quinoline-degrading bacteria biofilm: at lower concentration of quinoline, the bacteria formed large and developed aggregates; while at higher concentration, the bacterial aggregates became much smaller and evenly dispersive. Besides, the ratio of living cells of the biofilm formed at lower concentration was remarkably higher than that at higher concentration.