Subseafloor sediments are chronically resource-deficient habitats for life. Sediments beneath the subtropical gyres are especially impoverished due to the low particulate flux from the overlying water column. Nevertheless, they are populated with diverse microbial communities that manage to persist during millions of years of burial. In these environments, the principal source of energy supporting microbial metabolism is buried organic matter; hence, heterotrophic bacteria dominate such ecosystems. Electron acceptors (e.g., oxygen, nitrate, manganese oxides) are plentiful here, so these bacteria are believed to be growth limited by organic carbon. In this study of North Atlantic subtropical gyre sediments, we explored the possibility that nutrient (specifically phosphorus) availability might be more proximally limiting to microbial anabolism than energy supply. We developed and applied assays for key phosphorus acquisition enzymes (alkaline phosphatase and C-P lyase) and conducted radiobioassays testing the effects of phosphorus and acetate additions on protein and DNA synthesis rates. We found that the activity of both enzymes persists for at least 6.3 Ma, suggesting that phosphate monoesters and phosphonates represent important cellular resources over long periods. However, abiotic hydrolysis exceeded the alkaline phosphatase activity. Our bioassays suggested possible carbon/phosphorus co-limitation of protein synthesis but were inconclusive due to a strong stimulation of both protein and DNA synthesis by the filtered deep seawater used to prepare slurries. This unexpected result is consistent with nutrient limitation of growth, but further experiments will be necessary to determine whether the small concentration of phosphate in the natural seawater is the causative agent.