The current trend in sustainable development deals mainly with environmental management. There is a need for economically affordable, advanced treatment methods for the proper treatment and management of domestic wastewater containing excess nutrients (such as nitrogen and phosphorus) which can cause eutrophication. The reduction of the excess nutrient content of wastewater by appropriate technology is of much concern to the environmentalist. In the current study, a novel integrated anaerobic–anoxic–oxic activated sludge biofilm (A2O-AS-biofilm) reactor was designed and operated to improve the biological nutrient removal by varying reactor operating conditions such as carbon to nitrogen (C/N) ratio, suspended biomass, hydraulic retention time (HRT) and dissolved oxygen (DO). Based on various trials, it was seen that the A2O-AS-biofilm reactor achieved good removal efficiencies with regard to chemical oxygen demand (95.5%), total phosphorus (93.1%), ammonia nitrogen concentration (NH4+-N) (98%) and total nitrogen (80%) when the reactor was maintained at C/N ratio of 4, suspended biomass of 3 to 3.5 g/L, HRT of 10 h, and DO of 1.5 to 2.5 mg/L. Scanning electron microscopy (SEM) of suspended and attached biofilm showed a dense structure of coccus and bacillus bacteria with the diameter ranging from 0.3 to 1.2 μm. The Fourier transform infrared (FTIR) spectroscopy results indicated phosphorylated macromolecules and carbohydrates mix or bind with extracellular proteins in exopolysaccharides.