The role of electron donors (Fe2+ and Mn2+) in the autotrophic denitrification of contaminated groundwater by bacterial strain SY6 was characterized based on empirical laboratory-scale analysis. Strain SY6 can utilize Fe2+ more efficiently than Mn2+ as an electron donor. This study has shown that the highest nitrate removal ratio, observed with Fe2+ as the electron donor, was approximately 88.89%. An immobilized biological filter reactor was tested by using three levels of influent nitrate (10, 30, and 50 mg/L), three pH levels (6, 7, and 8), and three levels of hydraulic retention time (HRT; 6, 8, and 12 h), respectively. An optimal nitrate removal ratio of about 95% was achieved at pH 6.0 using a nitrate concentration of 50 mg/L and HRT of 12 h with Fe2+ as an electron donor. The study showed that 90% of Fe2+ and 75.52% removal of Mn2+ were achieved at pH 8.0 with a nitrate concentration of 50 mg/L and a HRT of 12 h. Removal ratio of Fe2+ and Mn2+ is higher with higher influent nitrate and HRT. A weakly alkaline environment assisted the removal of Fe2+ and Mn2+.