Abstract Bacteria and fungi are core microorganisms in diverse ecosystems, and their cross‐kingdom interactions are considered key determinants of microbiome structure and ecosystem functioning. However, how bacterial‐fungal interactions mediate soil organic carbon (SOC) dynamics remains largely unexplored in the context of artificial forest ecosystems. Here, we characterised soil bacterial and fungal communities in four successive planting of Eucalyptus and compared them to a neighbouring evergreen broadleaf forest. Carbon (C) mineralisation combined with five C‐degrading enzymatic activities was investigated to determine the effects of successive planting of Eucalyptus on SOC dynamics. Our results indicated that successive planting of Eucalyptus significantly altered the diversity and structure of soil bacterial and fungal communities and increased the negative bacterial‐fungal associations. The bacterial diversity significantly decreased in all Eucalyptus plantations compared to the evergreen forest, while the fungal diversity showed the opposite trend. The ratio of negative bacterial‐fungal associations increased with successive planting of Eucalyptus due to the decrease in SOC, ammonia nitrogen (NH₄⁺‐N), nitrate nitrogen (NO₃⁻‐N) and available phosphorus (AP). Structural equation modelling indicated that the potential cross‐kingdom competition, based on the ratio of negative bacterial‐fungal correlations, was significantly negatively associated with the diversity of total bacteria and keystone bacteria, thereby increasing C‐degrading enzymatic activities and C mineralisation. Synthesis and applications: Our results highlight the regulatory role of the negative bacterial‐fungal association in enhancing the correlation between bacterial diversity and C mineralisation. This suggests that promoting short‐term successive planting in the management of Eucalyptus plantations can mitigate the impact of this association on SOC decomposition. Taken together, our study advances the understanding of bacterial‐fungal negative associations to mediate carbon mineralisation in Eucalyptus plantations, giving us a new insight into SOC cycling dynamics in artificial forests.