A rare finding of early mature undersaturated oils with low gas/oil ratios enables us to document secondary microbial methane generation during very slight biodegradation in a deep hot reservoir in the ultradeep-water of the Gulf of Mexico. In three studied gas samples, methane is enriched in 13C (δ13C is from -63‰ to -64‰) relative to pure thermogenic methane (estimated δ13C is from -71‰ to -67‰) and pure primary microbial methane (δ13C is -68‰). Carbon dioxide in gases has δ13C values that negatively correlate with δ13C values of pure thermogenic methane. Methane is unusually enriched in heavy isotope 2H relative to associated ethane. Some extracted oils are depleted in long-chain alkyl aromatics. These lines of geochemical evidence suggest anaerobic microbial degradation of oil and subsequent reduction of resulting carbon dioxide to methane. Although specific geobiological details of secondary microbial methane generation are unclear, this process may be partially responsible for charging some of the largest gas and gas hydrate fields in the world.