The conventional approaches for qualitative and quantitative assessment of microbial biomass in sediments, e.g., via quantitative polymerase chain reaction or intact polar lipids analysis, are rather tedious and time-consuming. Here we present a new, optimized and simple pyrolysis-gas chromatography/mass spectrometry protocol for rapid screening of sediment samples for the presence of microbial signals and quantification of bulk population. Analysis of microbial cultures of different bacterial and archaeal lineages as well as reference substances and model compounds provided molecular fingerprints for tracking microbial biomass. The diagnostic fingerprints consist of benzyl nitrile (derived from DNA and protein), 2-furanmethanol (from DNA and peptidoglycan), indole (from peptidoglycan and protein), phenol (from DNA, peptidoglycan and protein) and pyrrole (from peptidoglycan). Detection of microbial signals through the molecular fingerprints requires a cell density of at least 106 cells/g, whereas at least 107 cells/g is necessary for quantification. The pyrolysis method was applied to marine sediments from different depths (until ∼400 m below the seafloor at the Canterbury Basin) and with different organic carbon contents (0.11–6.99%).