This work investigates the performance of acclimatized biomass for denitrification of high strength nitrate waste (10,000 mg/L NO₃) from a nuclear industry in a continuous laboratory scale (32 L) and pilot scale reactor (330 L) operated over a period of 4 and 5 months, respectively. Effect of substrate fluctuations (mainly C/NO₃-N) on denitrification was studied in a laboratory scale reactor. Incomplete denitrification (95–96 %) was observed at low C/NO₃-N (≤2), whereas at high C/NO₃-N (≥2.25) led to ammonia formation. Ammonia production increased from 1 to 9 % with an increase in C/NO₃-N from 2.25 to 6. Complete denitrification and no ammonia formation were observed at an optimum C/NO₃-N of 2.0. Microbiological studies showed decrease in denitrifiers and increase in nitrite-oxidizing bacteria and ammonia-oxidizing bacteria at high C/NO₃-N (≥2.25). Pilot scale studies were carried out with optimum C/NO₃-N, and sustainability of the process was checked on the pilot scale for 5 months.