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<p>The coexistence of many <span class="inline-formula">N₂O</span> production pathways in soil hampers differentiation of microbial pathways. The question of whether fungi are significant contributors to soil emissions of the greenhouse gas nitrous oxide (<span class="inline-formula">N₂O</span>) from denitrification has not yet been resolved. Here, three approaches to independently investigate the fungal fraction contributing to <span class="inline-formula">N₂O</span> from denitrification were used simultaneously for, as far as we know, the first time (modified substrate-induced respiration with selective inhibition (SIRIN) approach and two isotopic approaches, i.e. end-member mixing approach (IEM) using the <span class="inline-formula">¹⁵N</span> site preference of <span class="inline-formula">N₂O</span> produced (<span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M8" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msub><mi mathvariant="normal">SP</mi><mrow><msub><mi mathvariant="normal">N</mi><mn mathvariant="normal">2</mn></msub><mi mathvariant="normal">O</mi></mrow></msub></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="33pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="f08b96753db2216d06e87dc70dc009c1"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-4629-2021-ie00001.svg" width="33pt" height="15pt" src="bg-18-4629-2021-ie00001.png"/></svg:svg></span></span>) and the SP/<span class="inline-formula"><i>δ</i>¹⁸O</span> mapping approach (SP/<span class="inline-formula"><i>δ</i>¹⁸O</span> Map)). This enabled a comparison of methods and a quantification of the importance of fungal denitrification in soil.</p> <p>Three soils were incubated in four treatments of the SIRIN approach under anaerobic conditions to promote denitrification. While one treatment without microbial inhibition served as a control, the other three treatments were amended with inhibitors to selectively inhibit bacterial, fungal, or bacterial and fungal growth. These treatments were performed in three variants. In one variant, the <span class="inline-formula">¹⁵N</span> tracer technique was used to estimate the effect of <span class="inline-formula">N₂O</span> reduction on the <span class="inline-formula">N₂O</span> produced, while two other variants were performed under natural isotopic conditions with and without acetylene.</p> <p>All three approaches revealed a small contribution of fungal denitrification to <span class="inline-formula">N₂O</span> fluxes (<span class="inline-formula"><i>f</i>_FD</span>) under anaerobic conditions in the soils tested. Quantifying the fungal fraction with modified SIRIN was not successful due to large amounts of uninhibited <span class="inline-formula">N₂O</span> production. In only one soil could <span class="inline-formula"><i>f</i>_FD</span> be estimated using modified SIRIN, and this resulted in 28 <span class="inline-formula">±</span> 9 %, which was possibly an overestimation, since results obtained by IEM and SP/<span class="inline-formula"><i>δ</i>¹⁸O</span> Map for this soil resulted in <span class="inline-formula"><i>f</i>_FD</span> of below 15 % and 20 %, respectively. As a consequence of the unsuccessful SIRIN approach, estimation of fungal <span class="inline-formula"><math xmlns="http://www.w3.org/1998/Math/MathML" id="M21" display="inline" overflow="scroll" dspmath="mathml"><mrow class="chem"><msub><mi mathvariant="normal">SP</mi><mrow><msub><mi mathvariant="normal">N</mi><mn mathvariant="normal">2</mn></msub><mi mathvariant="normal">O</mi></mrow></msub></mrow></math><span><svg:svg xmlns:svg="http://www.w3.org/2000/svg" width="33pt" height="15pt" class="svg-formula" dspmath="mathimg" md5hash="634a5d3b3d6eb040b50398ae783f99b7"><svg:image xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="bg-18-4629-2021-ie00002.svg" width="33pt" height="15pt" src="bg-18-4629-2021-ie00002.png"/></svg:svg></span></span> values was impossible.</p> <p>While all successful methods consistently suggested a small or missing fungal contribution, further studies with stimulated fungal <span class="inline-formula">N₂O</span> fluxes by adding fungal C substrates and an improved modified SIRIN approach, including alternative inhibitors, are needed to better cross-validate the methods.</p>