The influence of ammonia nitrogen and natural organic matter on the performance of filter column for iron and manganese removal was analyzed, and the thickness of filter layer required by the filter column was explored. A pilot-scale biofilter column was constructed to remove iron, manganese, ammonia nitrogen, and natural organic matter (TFe 4.7-5.4 mg/L, Mn(Ⅱ) 1.1-1.3 mg/L, NH+₄-N 1.4-1.8 mg/L, COD_Mn 5.9-7.4 mg/L) from simulated groundwater under the conditions of water temperature of 14-17 ℃. The filter column used in the test was a mature biofilter column for iron and manganese removal. The startup of purifying ammonia nitrogen and natural organic matter in the same layer by means of cyclic inoculation and natural membrane hanging lasted for 79 d. Results show that the presence of ammonia nitrogen caused the Mn(Ⅱ) removal section of the filter column to move down, but it had no significant effect on the removal of iron and manganese by filter column. Fe(Ⅱ), Mn(Ⅱ), and ammonia nitrogen could be removed simultaneously under the condition of sufficient dissolved oxygen (DO). The presence of natural organic matter reduced the ability of the filter layer to purify iron and manganese. The efficient removal section for iron and manganese moved down, and the required filter thickness increased significantly. Natural organic matter could promote the removal of ammonia nitrogen to some extent. Proper backwashing had no effect on the purification performance of mature filter layer.