The return of agricultural crop residues are vital to maintain or even enhance soil fertility. However, the influence of application rate of crop residues on denitrification and its related gaseous N emissions is not fully understood. We conducted a fully robotized continuous flow incubation experiment using a Helium/Oxygen atmosphere over 30 days to examine the effect of maize straw application rate on: i) the rate of denitrification, ii) denitrification product stoichiometry N2O/(N2O+N2), and iii) the contribution of fungal denitrification to N2O fluxes. Five treatments were established using sieved, repacked sandy textured soil; i) non-amended control, ii) nitrate only, iii) low rate of straw + nitrate, iv) medium rate of straw + nitrate, and iv) high rate of straw + nitrate (n = 3). We simultaneously measured NO, N2O as well as direct N2 emissions and used the N2O 15N site preference signatures of soil-emitted N2O to distinguish N2O production from fungal and bacterial denitrification. Uniquely, soil NO3 − measurements were also made throughout the incubation. Emissions of N2O during the initial phase of the experiment (0–13 days) increased almost linearly with increasing rate of straw incorporation and with (almost) no N2 production. However, the rate of straw amendment was negatively correlated with N2O, but positively correlated with N2 fluxes later in the experimental period (13–30 days). Soil NO3 − content, in all treatments, was identified as the main factor responsible for the shift from N2O production to N2O reduction. Straw amendment immediately lowered the proportion of N2O from bacterial denitrification, thus implying that more of the N2O emitted was derived from fungi (18 ± 0.7% in control and up to 40 ± 3.0% in high straw treatments during the first 13 days). However, after day 15 when soil NO3 − content decreased to <40 mg NO3 −-N kg−1 soil, the N2O 15N site preference values of the N2O produced in the medium straw rate treatment showed a sharp declining trend 15 days after onset of experiment thereby indicating a clear shift towards a more dominant bacterial source of N2O. Our study singularly highlights the complex interrelationship between soil NO3 − kinetics, crop residue incorporation, fungal denitrification and N2O/(N2O + N2) ratio. Overall we found that the effect of crop residue applications on soil N2O and N2 emissions depends mainly on soil NO3 − content, as NO3 − was the primary regulator of the N2O/(N2O + N2) product ratio of denitrification. Furthermore, the application of straw residue enhanced fungal denitrification, but only when the soil NO3 − content was sufficient to supply enough electron acceptors to the denitrifiers.