Vegetables can be treated with biofertilizers as an alternative to chemical fertilizers because of their low toxicity. We investigated the effects of foliar spraying of Azotobacter under different levels of nitrogen (100, 150 and 200 mg/L in nutrient solution) on the growth, nutritional value, nitrate accumulation and antioxidant enzyme activities of hydroponically grown lettuce. The experiment was laid out in a completely randomized design with four replicates in a factorial combination. Plants treated with Azotobacter and 200 mg/L nitrogen had greater leaf area and photosynthetic pigments than plants treated with 200 mg/L nitrogen without spraying with Azotobacter. Increasing nitrogen levels increased leaf number, fresh and dry weights, leaf area and nitrate accumulation in lettuce plants. Peroxidase (POD) activity increased by 95.4% at a nitrogen level of 200 mg/L compared to a nitrogen level of 100 mg/L. Ascorbate peroxidase (APX) activity and leaf phosphorous (P) and potassium (K) concentrations were the highest in plants treated with a nitrogen source of 100 mg/L without foliar application of Azotobacter. As nitrogen levels increased in all treatments, nitrate reductase (NR) activity decreased and reached a minimum at the 200 mg/L nitrogen level. In general, foliar application of Azotobacter sp. can be used to promote plant growth and reduce nitrate accumulation in lettuce.