AbstractMicrobial necromass carbon (MNC) is a major contributor to soil organic carbon (SOC) sequestration. Fertilization combined with plastic film mulching, as an intensive agricultural practice to increase crop yields, affects soil microbial growth and metabolism. Nevertheless, how fertilization combined with mulching affects SOC sequestration by mediating MNC dynamics remains elusive. Here, the mulching and no‐mulching sub‐treatments were set under three fertilization treatments (no fertilization, NF; inorganic fertilization, IF; manure, MF), and a 900‐day field incubation experiment using polyvinyl chloride containers was conducted in the buffer zone of NF treatment. We investigated the effects of fertilization combined with mulching on MNC composition (including fungal necromass carbon, FNC; and bacterial necromass carbon, BNC) and qualified their contributions to SOC sequestration. The MF treatment with/without mulching significantly increased the contents of MNC, FNC, BNC, and SOC by 97%–122%, 81%–94%, 152%–210%, and 60%–70% compared with the NF treatment without mulching over 900 days, respectively. The MNC content had a strongly positive correlation with particulate organic carbon and microbial biomass C (p < 0.01). During the incubation stage, the proportion of MNC in SOC was higher in the IF (37%–42%) and MF (40%–44%) treatments with/without mulching than that in the NF treatment with/without mulching (31%–35%). On day 900, mulching significantly decreased the MNC content by 8.7% and 7.8%, and decreased the proportion of MNC in SOC by 5.1% and 5.8% under the NF and IF treatments, respectively. In contrast, mulching did not significantly affect the MNC and SOC contents, but significantly increased the FNC content by 4.8% under the MF treatment on day 900. Mulching significantly decreased the proportion of BNC in SOC regardless of fertilization, and increased the proportion of FNC in SOC under the MF treatment on day 900. Overall, our findings suggest that mulching under manure application maintains SOC sequestration by promoting FNC retention.