Fossilized microorganisms preserved as fluid inclusions are found in barite–silica-Mn oxide veins in the marine rift basin-related Quaternary Mn-Ba deposit of Vani, Milos. Basin fill consists of 35-50 m thick sequence of glauconitic sediments sandwiched between volcaniclastic sandy tuffs, and bedding-parallel barite–Mn oxide(–silica) horizons, pebble horizons, and massive gravel. Exhalative barite-rich deposits characteristic of sea-floor venting, such as white smoker (sulphate) structures in glauconitic sediments, feeder veins, bedding-conformable horizons, and extensive microbial mat- related structures in sandy tuffs, were recognized. The feeder veins host the microfossils and consist chiefly of banded barite and minor colloform quartz, Fe-oxyhydroxides, and hollandite-group minerals and MnO2 phases, and display epithermal textures characteristic of open-space precipitation. Curvilinear, branched filamentous microfossils with distinct segmentation of septa and a turgid appearance of knob-like outgrowths occur associated with spheroidal spore-like microfossils and small twisted microstructures. Both filamentous and spheroidal microstructures are filled with aqueous (liquid ± vapour) and/or hydrocarbon phases. Oil and solid hydrocarbons in the fluid inclusions may represent decomposed biological material. Chitin was detected by the pigment Wheat Germ Agglutinin conjugated with Fluorescein Isothiocyanate (WGA-FITC) in some of the microfossils, indicating that they are fossilized fungi; a fungal interpretation is further supported by microfossil morphology. Smaller, often twisted filamentous microfossils with a simpler morphology in which chitin was not detected probably represent fossilized prokaryotes and, if so, prokaryotes and eukaryotes co-existed in the geothermal system of Vani. Fluid inclusion microthermometry shows that microfossils were trapped at temperatures of ~100°C in boiling water, probably evolved seawater. Preservation of microfossils occurred at shallow sub-marine conditions of <10 m depth. Our results show that fluid inclusions may contain valuable palaeobiological information and can be used both for establishing biogenicity but also for the reconstruction of the palaeoenvironment of fossilized microorganisms.