Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) with a bismuth cluster primary ion source was used for analysing microbial lipid biomarkers in 10-µm-thick microscopic cryosections of methanotrophic microbial mats from the Black Sea. Without further sample preparation, archaeal isopranyl glycerol di- and tetraether core lipids, together with their intact diglycoside (gentiobiosyl-) derivatives, were simultaneously identified by exact mass determination. Utilizing the imaging capability of ToF-SIMS, the spatial distributions of these biomarkers were mapped at a lateral resolution of < 5 µm in 500 × 500 µm2 areas on the mat sections. Using cluster projectiles in the burst alignment mode, it was possible to reach a lateral resolution of 1 µm on an area of 233 × 233 µm, thus approaching the typical size of microbial cells. The mappings showed different ‘provenances’ within the sections that are distinguished by individual lipid fingerprints, namely (A) the diethers archaeol and hydroxyarchaeol co-occurring with glycerol dialkyl glycerol tetraethers (GDGT), (B) hydroxyarchaeol and dihydroxyarchaeol, and (C) GDGT and gentiobiosyl-GDGT. Because ToF-SIMS is a virtually nondestructive technique affecting only the outermost layers of the sample surface (typically 10–100 nm), it was possible to further examine the studied areas using conventional microscopy, and associate the individual lipid patterns with specific morphological traits. This showed that provenance (B) was frequently associated with irregular, methane-derived CaCO3 crystallites, whereas provenance (C) revealed a population of fluorescent, filamentous microorganisms showing the morphology of known methanotrophic ANME-1 archaea. The direct coupling of imaging mass spectrometry with microscopic techniques reveals interesting perspectives for the in-situ study of lipids in geobiology, microbial ecology, and organic geochemistry. After further developing protocols for handling different kinds of environmental samples, ToF-SIMS could be used as a tool to attack many challenging problems in these fields, such as the attribution of biological source(s) to particular biomarkers in question, or the high-resolution tracking of biogeochemical processes in modern and ancient natural environments.