Nalidixic acid is against a variety of microorganisms, it is against with Escherichia coli , Pasteurella spp. , Klebsiella pneuiiioniae , Aerobacter aeroyenes , Proteus spp. , Salmonella spp. , Shigella spp. and Brucella spp. with MIC values of 5.0-12.5 μg/ml, 0.5-2.5 μg/ml, 0.8-25.0 μg/ml, 1.0-25.0 μg/ml, 1.25-30.0 μg/ml, 8-3.2 μg/ml, and 7.5-10.0 μg/ml, respectively.

The in vivo activity of Nalidixic acid is most pronounced against Gram-negative bacteria, while Gram-positive organisms are generally more resistant. Maximal activity is observed against systemic infections caused by E. coli , A. aerobacter , Proteus mirabilis , Shigella fkxneri , the ED ₅₀ values are 25 mg/kg, 60 mg/kg, 50 mg/kg, and 62 mg/kg, respectively. The acute toxicity (LD ₅₀ ) of Nalidixic acid in mice following oral and parenteral administration is: oral, 3300 mg/kg; intravenous, 176 mg/kg; and subcutaneous, 500 mg/kg.