Bacteria and fungi have been shown to produce nitrous oxide (NO) during denitrification, but their contribution after nitrate (NO) application to soil is not clearly established. In a microcosm experiment, the relative contribution of bacteria and fungi to NO and carbon dioxide (CO) production by four contrasting soils representing different land uses after KNO addition was studied. The soils were daily wetted to 80% water-filled pore space (WFPS) and kept under greenhouse conditions for 10 days. The fungicide cycloheximide and the bactericide streptomycin were used to determine the possible microbial origin of the NO and CO emissions. Non-target effects of the antibiotics on the emission of NO and CO were evaluated using the inhibitor additivity ratio (IAR). The abundance of the bacterial and fungal communities was estimated by quantitative PCR (qPCR) of the bacterial 16S rRNA gene and the fungal internal transcribed spacer (ITS) region, respectively. The gene copy number of bacterial denitrifiers was calculated after quantification of the nirK, nirS, norB, nosZI and nosZII genes. After 10 d, regardless of the soil type, the cumulative NO emission from the soils treated with cycloheximide or streptomycin were similar. In all the four soils, NO fluxes were greater (on average 1.8 ± 0.3 times) in soils amended with the fungicide than with the bactericide during incubation for the first 48–96 h. Greater NO emissions (on average 1.7 ± 0.2 times) were detected in soils where bacteria were inhibited in comparison to those treated with the fungicide from 96 to 240 h. On average, 68.5% of the total CO emitted during the 10-d incubation period was produced in soils treated with the fungicide and 31.5% in those treated with the bactericide. The greater contribution of bacteria to the production of NO than fungi during the first 48–96 h was possibly due to a faster used of nitrate. Variations in the abundance of bacterial 16S rRNA genes, the ITS region, and the nirK, nirS, norB and nosZI bacterial denitrification genes indicated that the antibiotics used to prevent the growth of bacteria and fungi were effective during incubation. These results suggest that both bacteria and fungi should be considered when designing and applying greenhouse gas mitigation strategies in soils and that their relative contribution to produce NO and CO can vary with time and nitrate availability. This study was supported by the ERDF-cofinanced grant AGL2017–85676R from Ministerio de Economía, Industria y Competitividad. ACH thanks the Federation of European Microbiological Societies (FEMS) Research Grant 2017–2 program (FEMS-RG-2017-0067) and Ministerio de Educación Cultura y Deporte (MECD) for grant FPU 2014/01633. The authors would also like to acknowledge the UK Biotechnology and Biological Sciences Research Council (BBSRC) funded projects BBS/E/C/000I0310, BBS/E/C/000I0320 and BBS/E/C/000J0100 and UK Natural Environment Research Council (NERC) funded projects NE/M013847/1 and NE/M015351/1.