Herein, the progress of nitrate removal by a heterotrophic culture in a batch reactor and continuous-flow fixed-biofilm reactor was examined. Two batch experiments for nitrate reduction with acetate degradation using 250 mL batch reactors with acclimated denitrifying biomass were conducted. The experimental results indicated that the nitrate was completely reduced; however, the acetate remained at a concentration of 280 mg/L from initial nitrate concentration of 100 mg/L. However, the acetate was fully biodegraded by the denitrifying biomass at an initial nitrate concentration of 300 mg/L. To evaluate the biokinetic parameters, the concentration data of nitrate, nitrite, acetate, and denitrifying biomass from the batch kinetic experiments were compared with those of the batch kinetic model system. A continuous-flow fixed-biofilm reactor was used to verify the kinetic biofilm model. The removal efficiency of nitrate in the fixed-biofilm reactor at the steady state was 98.4% accompanied with 90.5% acetate consumption. The experimental results agreed satisfactorily with the model predictions. The modeling and experimental approaches used in this study could be applied in the design of a pilot-scale, or full-scale, fixed-biofilm reactor for nitrate removal in water and wastewater treatment plants.