Wetland and permafrost soils contain some of Earth's largest reservoirs of organic carbon, and these stores are threatened by rapid warming across the Arctic. Nearly half of northern wetlands are affected by permafrost. As these ecosystems warm, the cycling of dissolved organic matter (DOM) and the opportunities for microbial degradation are changing. This is particularly evident as the relationship between wetland and permafrost DOM dynamics evolves, especially with the introduction of permafrost-derived DOM into wetland environments. Thus, understanding the interplay of DOM composition and microbial communities from wetlands and permafrost is critical to predicting the impact of released carbon on global carbon cycling. As little is understood about the interactions between wetland active layer and permafrost-derived sources as they intermingle, we conducted experimental bioincubations of mixtures of DOM and microbial communities from two fen wetland depths (shallow: 0–15 cm, and deep: 15–30 cm) and two ages of permafrost soil (Holocene and Pleistocene). We found that the source of microbial inoculum was not a significant driver of dissolved organic carbon (DOC) degradation across treatments; rather, DOM source and specifically, DOM molecular composition, controlled the rate of DOC loss over 100 days of bioincubations. DOC loss across all treatments was negatively correlated with modified aromaticity index, O/C, and the relative abundance of condensed aromatic and polyphenolic formula, and positively correlated with H/C and the relative abundance of aliphatic and peptide-like formula. Pleistocene permafrost-derived DOC exhibited ∼70% loss during the bioincubation driven by its initial molecular-level composition, highlighting its high bioavailability irrespective of microbial source.