Pilot-scale biofilters treated with real groundwater were established to systemically investigate the interactions between ammonia, iron and manganese removal. When both of ammonia and manganese in influent were about 1 mg/L, they were quickly removed; but when manganese was above 3 mg/L, ammonia removal was affected significantly. When total iron was above 5 mg/L, the oxidization rate of ammonia decreased significantly. In addition, manganese and iron removal were not affected by ammonia, when ammonia was lower than 2.5 mg/L. Fe2+ could react with manganese oxides in the biofilter, thus manganese removal could only take place after Fe2+ was completely oxidized. When total iron, manganese and ammonia in influent were 11.27 mg/L, 1.20 mg/L and 1.27 mg/L, respectively, iron, ammonia and manganese were mainly removed in 0–0.3 m, 0–0.4 m and 0.2–0.8 m of the filter depth, respectively, and nitrite oxidizing bacteria were presented in 0–0.3 m of the filter depth. The pH decreased along the filter depth with the oxidation of ammonia, iron and manganese, while oxidation reduction potential increased. The results of this study were useful for the optimization and design of biofilters.