Soil amended with biochar is expected to produce less nitrous oxide (N₂O), although this may depend on nitrate (NO₃-N) availability. Our objective was to determine how pine wood biochar, slow pyrolyzed at 500 °C, affects N₂O production in soil having different denitrification potentials with variable NO₃-N concentrations under controlled laboratory conditions. Sandy loam surface soils (0–30 cm, pH 5.7) and sandy clay loam subsurface soils (40–60 cm, pH 5.6) were amended with four biochar rates (0, 10, 20, and 30 g·kg⁻¹), two nitrogen fertilizer rates (0 and 100 mg·kg⁻¹ NO₃-N) and two acetylene levels (0% and 10% headspace), arranged as a full factorial. Soil moisture content was adjusted to 80% water-filled pore space, and flasks were incubated at 20 °C for 30 h. Headspace gas was collected from each flask at 25, 26, 28, and 30 h. There was a significant reduction in N₂O production with an increasing rate of biochar in the surface soil but not in the subsurface soil. On average, less N₂O was produced in the subsurface soil than in the surface soil. As the NO₃-N concentration was not a limiting factor for denitrification, the most likely explanation was that denitrifier activity was influenced by the availability of soluble organic carbon in the soil–biochar mixtures. We recommend further study of the coupled carbon–nitrogen transformations during denitrification to understand how biochar influences soil N₂O production in sandy loam soils.