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Acetylene (C2H2) is often employed to assess soil total denitrification (N2O + N2) due to its ease of implementation. However, this technique underestimates soil denitrification in soils with low nutrient contents, particularly those supporting grain yields. To our knowledge, there are limited studies that have specifically investigated the impact of C2H2 on nutrient-rich vegetable soils, especially concerning the emissions of N2 and N2O and the nitrogenous gas product ratio (i.e., N2O/(N2O + N2)). In this study, we conducted both anoxic and oxic incubations at various C2H2 concentrations (0%, 0.01%, and 10%, v/v) and utilized a robotized sampling and analysis system to quantify soil N2, N2O, and CO2 emissions. Our findings revealed that the cumulative N2O production in soil treated with 10%C2H2 was significantly lower than that in soil treated with 0.01%C2H2 and soil without C2H2. Contrarily, high concentrations of C2H2 (10%, v/v) led to increased N2 production. Similar trends were observed under oxic conditions, where 10%C2H2 concentration did not enhance N2O production but markedly increased N2 and CO2 emissions. Moreover, the N2O/(N2O + N2) product ratio was notably higher in soils treated with 0%C2H2 compared to the 10%C2H2 treatment under anoxic conditions. These findings indicate that high concentrations of acetylene could facilitate the reduction of N2O to N2 and lead to underestimated soil total denitrification in vegetable soil, regardless of anoxic or oxic conditions. This discovery underscores the drawbacks when employing high concentrations of acetylene to evaluate actual total denitrification in intensive greenhouse vegetable soils, highlighting the necessity for further investigation into alternative methodologies.