This study compared the performance of heterotrophic and hydrogenotrophic denitrification for nitrate removal from well water using two 1-L batch reactors operated under anoxic conditions for 48 h. After a one-month sludge enrichment period, dextrose was used as the carbon source for heterotrophic denitrification, while hydrogen and carbon dioxide gases supported hydrogenotrophic denitrification. Both systems were evaluated for removing 50 mg/L nitrate-nitrogen. The heterotrophic process reduced nitrate-nitrogen to below 10 mg/L within 12 h, whereas the hydrogenotrophic system required approximately 24 h to achieve the same concentration. However, nitrite accumulated to 20 mg/L in the heterotrophic reactor before declining to an average of 4 mg/L, which remained above the recommended standard. The heterotrophic system also produced a residual chemical oxygen demand (COD) of 95 mg/L and a biomass concentration of 0.09 g/L. In contrast, the hydrogenotrophic reactor maintained nitrite below 3 mg/L, residual COD below 15 mg/L, and biomass at 0.05 g/L at pH 7–8. Although heterotrophic denitrification achieved faster nitrate removal, hydrogenotrophic denitrification produced higher-quality effluent because of its lower biomass production, lower residual COD, and minimal nitrite accumulation. Nevertheless, optimization of hydrogen supply and phosphorus supplementation is required to improve process efficiency and support practical groundwater treatment.