Acidic red soils are critical agricultural land resources in southern China, where straw incorporation and green manure incorporation are widely adopted as organic fertilization practices. However, the long-term effects of different organic material inputs on the abundances of bacterial and fungal denitrification functional genes and their depth-dependent responses remain poorly understood. A long-term field experiment established in 2010 in Dongxiang, Jiangxi Province, China, was used to investigate four fertilization treatments: CK (no fertilizer), NPK (mineral fertilizer), NPKS (mineral fertilizer plus straw incorporation), and NPKG (mineral fertilizer plus green manure incorporation). Soil samples were collected from the 0-15 cm and 15-30 cm layers in 2023 and 2025. The abundances of the bacterial denitrification functional genes nirK and nirS and the fungal denitrification functional gene FnirK were quantified by quantitative real-time PCR (qPCR), and correlation analyses were performed to clarify the major environmental drivers. Compared with the NPK treatment, NPKS consistently increased nirK abundance in the surface soil in both 2023 and 2025, and significantly increased nirS and FnirK abundances in 2025 and 2023, respectively. In contrast, NPKG significantly increased nirK abundance only in 2023 and showed no significant effects on nirS or FnirK, indicating a weaker stimulatory effect than straw incorporation. None of the fertilization treatments significantly affected denitrification functional gene abundances in the subsurface soil. Both nirK and nirS exhibited clear enrichment in the surface soil, whereas FnirK abundance did not differ significantly between soil depths. Correlation analysis showed that nirS abundance was primarily associated with soil pH and nutrient availability, while the relationships of nirK and FnirK with environmental factors varied markedly between soil depths. Long-term organic material inputs significantly altered the abundances of denitrification functional genes in acidic red soils, with responses strongly dependent on both material type and soil depth. Straw incorporation exerted a stronger enhancement effect on the abundance of denitrification functional genes, suggesting that it may have higher denitrification functional potential. In contrast, green manure incorporation may have better environmental benefits, although this inference requires further validation via in situ N2O emission measurements.