The late stage of petroleum hydrocarbon releases to the subsurface is an evolving but largely unexplored concept. Herein, transmissive aquifer zones with little remaining petroleum liquids become attenuation zones for dissolved organic species released from low-permeability (low-k) zones via back diffusion. To address the knowledge gaps surrounding these subsurface zones, we explored a 40-year-old depleted petroleum body at a former refinery through high-resolution chemical and biomolecular analyses of a cryogenically collected soil core. 16S rRNA gene transcript-based analyses of active microbial communities uncovered predominately aerobic bacteria in the transmissive zone in contrast to anaerobic fermenting bacteria and methanogenic archaea in the low-k zone. Unexpectedly, Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) analyses revealed a substantially higher degree of oxygenation in the petroleum biodegradation metabolites from the anoxic low-k zone compared to species in the oxic transmissive zone. Likely, a small diffusive influx of molecular oxygen enables limited aerobic metabolism in the low-k zone, while more abundant O 2 in the transmissive zone promotes rapid aerobic biodegradation of petroleum hydrocarbons without the accumulation of highly oxygenated intermediates. While much remains to be uncovered, our work is a critical first step toward enabling better-informed decision making regarding best management practices for late-stage petroleum hydrocarbonimpacted sites.