Fungal ability of N₂O production has been increasingly documented, yet its ecological importance and controlling factors remains unclear. In this study, the relative contribution of fungi versus bacteria to soil N₂O production was examined in five ecosystems, including conventional farming (CON), organic farming (ORG), integrated crop-livestock system (ICL), plantation forestry (PF), and abandoned arable field subjected to natural succession (SUCC). A laboratory microcosm experiment was conducted to measure soil N₂O production at 90% water-filled pore space (WFPS) in the antibiotic-free soil as well as soil amended with streptomycin, cycloheximide, or both. While soil N₂O production rates differed significantly among the five systems, fungal contribution accounted consistently for > 30% of total soil N₂O production. Fungal produced more N₂O than bacteria in PF, whereas both made comparable contributions in other four systems. To assess pH impacts on fungal and bacterial contributions to soil N₂O production, soil pH of CON and PF was adjusted to and maintained at 4 - 9 for one week and then soil N₂O production rates were measured with the aid of antibiotics. Both fungi and bacteria preferred neutral and slight alkaline conditions for N₂O production, but fungi contributed more compared to bacteria under acidic conditions. Thus, the ratio of fungal-to-bacterial contribution was negatively correlated with soil pH. Real-time PCR of 16S rDNA, 18S rDNA, norB, nirK, nirS, and nosZ also showed that fungal abundance was greater under acidic conditions, whereas bacterial and denitrifier ones were lower. Our result indicated that fungi could play an important role in soil N₂O production in diverse ecosystems and soil pH was a critical control factor.