Natural seeps contribute nearly half of the oil entering the coastal ocean. However, environmental fate studies generally monitor fewer than 5% of these petroleum compounds. Hence, the rates and relevance of physical, chemical, and biological weathering processes are unknown for the large majority of hydrocarbons, both released from natural seeps and also from human activities. To investigate the specific compositional changes occurring in petroleum during subsurface degradation and submarine seepage, we studied the natural oil seeps offshore Santa Barbara, California with comprehensive, two-dimensional gas chromatography (GC×GC). With this technique, we quantified changes in the molecular diversity and abundance of hydrocarbons between subsurface reservoirs, a proximal sea floor seep, and the sea surface overlying the seep. We also developed methods to apportion hydrocarbon mass losses due to biodegradation, dissolution, and evaporation, for hundreds of tracked compounds that ascended from the subsurface to the sea floor to the sea surface. The results provide the first quantitative evidence of broad metabolic specificity for anaerobic hydrocarbon degradation in the subsurface and reveal new trends of rapid hydrocarbon evaporation at the sea surface. This study establishes GC×GC as a powerful technique for differentiating biological and physical weathering processes of complex mixtures at a molecular level.