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Biological nitrogen removal is primarily conducted by bacteria and fungi rather than actinomycetes. However, accumulations of nitrite and hydroxylamine could significantly impair the biological nitrogen removal process. A strain of Streptomyces mediolani, termed EM-B2, was isolated from a cow dung fermentation biogas digester. The strain removed more than 99% of ammonium and 78% of total nitrogen in the presence of glucose and under environmental conditions of 30 °C, a carbon/nitrogen ratio of 15, 7.4 mg/L dissolved oxygen and a pH range of 7.5–9.0. Maximal removal rates were 2.29 mg/L/h for ammonium, 1.90 mg/L/h for nitrate and 2.01 mg/L/h for nitrite. The removal efficiencies of hydroxylamine and total nitrogen peaked at 81.48% and 60.38%, respectively. Notably, hydroxylamine and nitrite were never detected during the heterotrophic nitrification and aerobic denitrification. Nitrate rather than nitrite was accumulated from the process of hydroxylamine oxidation. These findings indicate that S. mediolani strain EM-B2 performs heterotrophic nitrification and aerobic denitrification, and can be used to remove hydroxylamine and nitrite from wastewater.