Significance Microbial cells are widespread in diverse deep subseafloor environments; however, the viability, growth, and ecophysiology of these low-abundance organisms are poorly understood. Using single-cell–targeted stable isotope probing incubations combined with nanometer-scale secondary ion mass spectrometry, we measured the metabolic activity and generation times of thermally adapted microorganisms within Miocene-aged coal and shale bed samples collected from 2 km below the seafloor during Integrated Ocean Drilling Program Expedition 337. Microorganisms from the shale and coal were capable of metabolizing methylated substrates, including methylamine and methanol, when incubated at their in situ temperature of 45 °C, but had exceedingly slow growth, with biomass generation times ranging from less than a year to hundreds of years as measured by the passive tracer deuterated water.