The dynamics and fate of terrestrial organic matter (OM) under elevated atmospheric CO2 and nitrogen (N) fertilization are important aspects of long-term carbon sequestration. Despite numerous studies, questions still remain as to whether the chemical composition of OM may alter with these environmental changes. In this study, we employed molecular-level methods to investigate the composition and degradation of various OM components in the forest floor (O horizon) and mineral soil (0–15 cm) from the Duke forest free air CO2 enrichment (FACE) experiment. We measured microbial responses to elevated CO2 and N fertilization in the mineral soil using phospholipid fatty acid (PLFA) profiles. Increased fresh carbon inputs into the forest floor under elevated CO2 were observed at the molecular-level by two degradation parameters of plant-derived steroids and cutin-derived compounds. The ratios of fungal to bacterial PLFAs and Gram-negative to Gram-positive bacterial PLFAs decreased in the mineral soil with N fertilization, indicating an altered soil microbial community composition. Moreover, the acid to aldehyde ratios of lignin-derived phenols increased with N fertilization, suggesting enhanced lignin degradation in the mineral soil. 1H nuclear magnetic resonance (NMR) spectra of soil humic substances revealed an enrichment of leaf-derived alkyl structures with both elevated CO2 and N fertilization. We suggest that microbial decomposition of SOM constituents such as lignin and hydrolysable lipids was promoted under both elevated CO2 and N fertilization, which led to the enrichment of plant-derived recalcitrant structures (such as alkyl carbon) in the soil.