Fungal communities play a key role in the decomposition of crop residues and affect soil organic carbon (SOC) dynamics. Conservation tillage enhances SOC sequestration and mitigate global climate change. However, the impact of long-term tillage practices on fungal community diversity and its relation to SOC stock remains unclear. The objectives of this study were to evaluate the relationship between extracellular enzyme activities and fungal community diversity and SOC stock under different tillage practices. A field experiment was conducted with four tillage practices: (i) no-tillage with straw removal (NT0), (ii) no-tillage with straw retention (NTSR, conservation tillage), (iii) plough tillage with straw retention (PTSR), and (iv) rotary tillage with straw retention (RTSR). The results showed that the SOC stock in NTSR was higher than other treatments in the 0-10 cm soil layer. Compared to NT0, NTSR significantly increased soil β-glucosidase, xylosidase, cellobiohydrolase, and chitinase activities at 0-10 cm soil depth (P < 0.05). However, different tillage methods with straw returning had no significant effects on enzyme activity at 0-10 cm soil depth. The observed species and Chao1 index of the fungal communities under NTSR were 22.8% and 32.1% lower than under RTSR in the 0-10 cm soil layer, respectively. The composition, structure, and co-occurrence network of fungal communities differed across tillage practices. A partial least squares path model (PLS-PM) analysis indicated that C-related enzymes were the most influential factors associated with SOC stock. Soil physicochemical properties and fungal communities affected extracellular enzyme activities. Overall, conservation tillage can promote surface SOC stock, which was associated with increased enzyme activity.