Polyethylene mulching film, which is widely utilized in arid and semi-arid agriculture, leaves residual pollution. A novel approach to addressing this issue is microbial degradation. To screen the strains that degrade polyethylene efficiently and clarify the effect of degrading strains on the turnover of soil organic carbon, a polyethylene-degrading fungus PF2, identified as Trichoderma asperellum, was isolated from long-time polyethylene-covered soil. Strain PF2 induced surface damage and ether bonds, ketone groups and other active functional groups in polyethylene, with 4.15% weight loss after 30 days, where laccase plays a key role in the degradation of polyethylene. When applied to soil, the Trichoderma-to-soil weight ratios were the following: B1: 1:100; B2: 1:200; B3: 1:300 and B4: 1:400. Trichoderma asperellum significantly increased the cumulative CO2 mineralization and soil organic carbon mineralization in the B1 and B2 treatments compared with the control (B0). The treatments B1, B3 and B4 increased the stable organic carbon content in soil. An increase in the soil organic carbon content was observed with the application of Trichoderma asperellum, ranging from 27.87% to 58.38%. A positive correlation between CO2 emissions and soil organic carbon was observed, with the soil carbon pool management index (CPMI) being most correlated with active organic carbon. Trichoderma treatments improved the CPMI, with B3 showing the most favorable carbon retention value. Thus, Trichoderma asperellum not only degrades polyethylene but also contributes to carbon sequestration and soil fertility when applied appropriately.