• Soil pH was a key driver of N2O emission and sources in acidic soils. • N2O emission was significantly positively associated with the ratio of ITS to 16S. • N2O was significantly correlated with bacterial and fungal community composition. • Fungi contributed to N2O in highly acidic tea plantations and vegetable fields. Acidic soil is a main source of global nitrous oxide (N2O) emissions. However, the mechanism behind the high N2O emissions from acidic soils remains a knowledge gap. The objective of this microcosm incubation study was to pin-point the microbial mechanisms involved in N2O production processes in acidic soils. For that purpose, the isotopic signatures and microbial community structure and composition of four soil samples were examined. The results showed that highly acid soils (pH = 3.51) emitted 89 times more N2O than alkaline soils (pH = 7.95) under the same nitrogen (N) inputs. Fungal denitrification caused high N2O emissions in acidic soils. ITS to 16S abundance ratio was positively correlated with cumulative N2O emissions from the tested soils. The highly acid soils (pH < 4.5) showed greater fungal nirK gene abundance and lower abundance of AOA-amoA, AOB-amoA, nirK, nosZ I and nosZ II genes. The unclassified Aspergillaceae fungi (63.65%) dominated the highly acidic soils and was the most strongly correlated genus with N2O emissions. These findings highlight that soil microbial community structures, denitrifying fungi in particular, shaped by low pH (pH < 4.5) lead to high N2O emissions from acidic soils.