Three horizontal subsurface flow constructed wetland prototypes were set up to identify and understand the role of microflora in nutrient removal under diverse operating conditions. Out of three setups, one setup served as a control (without plants), and the rest were planted with Typha domingensis. The setups were operated at two different hydraulic loading rates (5 cm/day and 16 cm/day) for two months each. Among 27 bacteria species isolated, 80% of nitrate-reducing bacteria were observed in control, and 50–77% of nitrate-reducing bacteria were observed in the plant setups. Presence of diverse denitrifying bacteria and soil organic carbon contributed to high Nitrate-N removal in the control at both HLRs. Similar Ammonium-N (29%) and Ortho-P removal (30%) efficiency was observed at both HLRs in the control setup. Processes such as chemical sorption and adsorption dominated the Ammonium-N and Ortho-P removal in the control setup. High average Ammonium-N removal efficiency of 89% and 52% was observed in plant setups at 5 cm/day and 16 cm/day HLR. At low HLR, Ammonium-N removal in plant setups was dominated by nutrient uptake. In the plant setups, 35% and 15% Ortho-P removal efficiency was observed at low HLR (5 cm/day) and high HLR (16 cm/day) respectively. Hydraulic Retention Time (HRT) limited the uptake of ortho-P, thereby allowing mineralised phosphorus to escape the system without being absorbed by the plants.