Soil microorganisms support multiple agroecosystem functions, but responses of the microbiome to prolonged agricultural land-use and underlying mechanisms remain poorly understood. In this study, upland soils in the Mollisol region of Northeast China were selected along a 100-year cultivation chronosequence to examine the effects of intensive agriculture on the diversity, composition and co-occurrence network of bacterial and fungal communities. To elucidate the determinants driving the dynamics and interactions of microbial communities, the researchers quantified the functional groups and bioavailable fractions of soil organic carbon, and estimated the microenvironments including soil texture and oxygen availability. Our findings revealed that fungal diversity and community stability decreased over cultivation time, while that of bacteria showed a less significant decline. The increased oxygen availability and decreased soil carbon supply, particularly polyphenols, jointly drove the dynamics in the composition and stability of the microbial communities. Crucially, these microbial responses were significantly regulated by interspecific associations. Phylotypes showing stronger responses to changes in oxygen availability and carbon supply demonstrated a higher level of connectivity within co-occurrence networks, signifying their potential role as pivotal “hubs” in transmitting the environmental influences across the entire community via interspecific associations. Moreover, the decline in fungal diversity over longer cultivation periods could liberate more survival niches for bacterial species through fungi-bacteria associations, thereby bolstering the stability of the bacterial community. Overall, this study accentuates the pivotal role of trophic associations in facilitating the oxygen and carbon-induced succession of belowground microbiota in long-term cultivated Mollisols. In view of the agricultural intensification propelled by the increasing demand for crop production, these findings suggest strategies integrating improvements in soil structure and carbon with microbial processes to ensure the sustainability of agroecosystem functions.