Soil organic matter (SOM) constitutes more than two-thirds of terrestrial carbon stocks yet there are many uncertainties about the fate of soil carbon reserves with global environmental change. Moisture, altered nutrient cycles, species shifts, growing season length or rising temperatures may alter forest primary productivity and the proportions of above and belowground biomass entering soil. We investigated SOM composition using molecular-level techniques after 20 years of detrital input and removal treatment (DIRT) at the Harvard Forest. SOM biomarkers (solvent extraction, base hydrolysis and cupric(II) oxide oxidation) and nuclear magnetic resonance (NMR) spectroscopy were used to quantify changes in SOM composition and microbial activity and community composition was assessed using phospholipid fatty acid (PLFA) analysis. Doubling aboveground litter inputs decreased soil carbon content, increased the degradation of labile SOM and enhanced the sequestration of aliphatic compounds in soil. The exclusion of belowground inputs resulted in a decrease in root-derived components and enhanced the degradation of stable SOM components such as leaf-derived aliphatic structures (cutin). The DIRT manipulations resulted in soil microbial community shifts that were attributed to the accelerated processing of specific SOM components. These results collectively reveal that a detailed molecular-level characterization of SOM can provide information on SOM compositional changes and transformations after 20 years of input manipulation in a temperate forest.