Improving soil organic matter (SOM) maintenance is crucial for terrestrial carbon (C) sequestration and ecosystem functioning. Conservation tillage favors SOM pool buildup; however, it remains unclear how the decomposition of heterogeneous components is manipulated by microbial substrate utilization strategy from the view of SOM stability. Here, a one-year microcosm incubation was conducted using surface soils developed under 12 years of conservation tillage (high-C soil) and maize residue removal (low-C soil). Temporal changes in lignin phenols, neutral sugars, and amino sugars in the soil were monitored along with microbial phospholipid fatty acids (PLFAs) and enzyme activities. Throughout incubation, lignin phenols declined more (20.8–26.3%) than the SOM (12.3–14.5%) and amino sugars (10.6–12.3%), highlighting the key role of plant debris in SOM mineralization, and complementarily, the greater contribution of microbial necromass to SOM stabilization. Moreover, the decomposition dynamics of neutral sugars and lignin were strongly influenced by C availability. In the low-C soil, these two types of compounds decomposed with similar temporal patterns and extents, and such substrate co-metabolism was dominantly mediated by actinomycetes. In contrast, in the high-C soil, a lower oxidases-to-carbohydrolases ratio regulated the sequential decomposition of labile neutral sugars followed by recalcitrant lignin. Such microbial substrate selectivity was associated with a shift in microbial community from bacterial dominance toward increased fungal contribution. Overall, our findings underscore the significant interplay between soil C availability and flexible microbial substrate utilization strategy in regulating decomposition of heterogeneous SOM components, as well as their distinct contributions in SOM turnover and stabilization.