Excessive anthropogenic nutrient inputs often lead to the degradation of wetland ecosystems and a decrease in carbon sink capacity. Microbial-derived carbon is increasingly recognized as an important precursor for organic carbon formation, which is controlled by the balance between microbial anabolic and catabolic processes. Shifts in microbial metabolic investment under nutrient load disturbance are key, but understudied, components of microbial-derived carbon turnover. Here, the roles of the distinct life-history traits and cooperation degree of key microbial assemblies in regulating microbial-derived carbon accumulation in a wetland receiving treated wastewater were firstly assessed by combining microbial biomarkers and genomic approaches. It was found that microbial-derived carbon was an important source of organic carbon in wetlands, and strongly associated with several microbial assemblies with specific trait strategies. Further analysis demonstrated that high growth yield strategists were mainly associated with microbial necromass accrual, while microbial biomass was more dominated by resource acquisition strategies in nutrient-imbalanced wetlands. A significant positive relationship between positive cohesion and microbial-derived carbon indicated that cooperative behavior among taxa promoted the production and accumulation of microbial-derived carbon. Moreover, resource stoichiometric balance, including C:N and C:P, was identified as an important driver of shifts in microbial metabolic investment strategies. The decreased C:N ratio led to a shift from resource acquisition strategies to high growth yield strategies for the microbial community, which facilitated microbial necromass accrual along the N-limited wetland, while the increased C:P ratio caused by excessive P deposition in sediments limits microbial cooperative growth to some extent. This study highlighted the importance of stoichiometric balance in mediating microbial growth metabolism and, in turn, enhancing the carbon sink capacity of wetlands.