Biologic activity is interpreted to have exerted control on Earth surface processes and the rock cycle since at least the early Archean. Evidence for the presence of life from this early phase only begins to be preserved in tangible fossils at about c. 3.5 Ga due to metamorphic overprint in older rocks. Characteristic depletions in 13C relative to 12C are instead widely recognized as “chemofossils” provided that subsequent disturbance or exchange can be ruled out. An important part of this record are inclusions of carbonaceous matter that are armoured by refractory minerals such as apatite or zircon as old as 4.1 Ga. To better constrain how such inclusions form and how they behave during protracted storage, we identified and investigated graphite inclusions in zircon from the early Phanerozoic S-type Rumburk granite in the Lusatian complex of the Bohemian Massif in eastern central Europe. The zircon host rock is a two-mica, cordierite-bearing peraluminous granite with a highly reduced mineral paragenesis, including wüstite, and it intruded into Neoproterozoic to Paleozoic metagraywacke and graphite-bearing metapelites. Graphite inclusions in zircon are rare: only five inclusions between 8 and 25 µm in diameter were identified in ~300 zircon crystals with opaque inclusions (the rest being ilmenite and rutile). Inclusions were trapped at the interface between inherited zircon cores and young overgrowths in voids left behind after metamict detrital zircon dissolved and reprecipitated prior to anatexis. Zircon fO2-barometry and Ti-in zircon thermometry revealed crystallization at FMQ for inherited cores, whereas c. 496 Ma overgrowths indicate low fO2 (FMQ -5) at ~700 °C. Heterogeneous O-isotopic compositions in interiors contrast with homogeneously elevated δ18O (+7.7‰) in rims indicating that the granite originated from melting metasediments. Structurally, graphite ranges from well-ordered to significantly disordered, possibly reflecting accumulation of lattice damage due to local irradiation by the zircon host. Carbon isotopic compositions determined by SIMS range from -45 to -8‰ (δ13C relative to Vienna Pee Dee Belemnite), with distributions for individual inclusions peaking at values of -43, -36, -34, -31, and -13‰. Significant heterogeneities over ~10–20 permil exist in individual graphite inclusions, typically skewed towards more positive values, in contrast to graphite references analysed under the same conditions which reproduced within ±2‰. Rayleigh fractionation models between 350 and 600 °C explain this range for graphite precipitation from a mixed CO2 > CH4 fluid with an initial bulk value of -32‰, consistent with fluids released from regional metasediments containing carbon of unambiguously biologic origin. Strong isotopic fractionation during graphite-forming reactions involving C-O-H fluids is thus to be expected for graphite in metamorphic and anatectic environments. If CO2 is the dominant C-bearing phase in the fluid, this fractionation leads to increasingly positive values in the residual fluid and graphite precipitating from it, so that the C isotopic composition of the source reservoir tends to be more negative than that of graphite. With their C-isotopic composition directly traceable to regional organic-rich metasediments, the graphitic inclusions in the Rumburk granite have thus preserved biogenic signatures over nearly 500 Ma.