The chemical and isotopic compositions of sedimentary organic matter (SOM) from two mid-slope sites of the northern Cascadia margin were investigated during Integrated Ocean Drilling Program (IODP) Expedition 311 to elucidate the organic matter origins and identify potential microbial contributions to SOM. Gas hydrate is present at both locations (IODP Sites U1327 and U1328), with distinct patterns of near-seafloor structural accumulations at the cold seep Site U1328 and deeper stratigraphic accumulations at the slope-basin Site U1327. Source characterization and evidence that some components of the organic matter have been diagenetically altered are determined from the concentrations and isotopic compositions of hydrocarbon biomarkers, total organic carbon (TOC), total nitrogen (TN) and total sulfur (TS). The carbon isotopic compositions of TOC (δ¹³C_TOC = −26 to −22‰) and long-chain n-alkanes (C₂₇, C₂₉ and C₃₁, δ¹³C = −34 to − 29‰) suggest the organic matter at both sites is a mixture of 1) terrestrial plants that employ the C3 photosynthetic pathway and 2) marine algae. In contrast, the δ¹⁵N_TN values of the bulk sediment (+ 4 to + 8‰) are consistent with a predominantly marine source, but these values most likely have been modified during microbial organic matter degradation. The δ¹³C values of archaeal biomarker pentamethylicosane (PMI) (− 46.4‰) and bacterial-sourced hopenes, diploptene and hop-21-ene (− 40.9 to − 34.7‰) indicate a partial contribution from methane carbon or a chemoautotrophic pathway. Our multi-isotope and biomarker-based conclusions are consistent with previous studies, based only on the elemental composition of bulk sediments, that suggested a mixed marine-terrestrial organic matter origin for these mid-slope sites of the northern Cascadia margin.