Evidence of bioalteration of natural basaltic rocks, presently receiving much attention, has so far been restricted to in situ oceanic crust and ophiolites in which fresh glass is still present. Here we present evidence of preserved bio-signatures in the chilled margin of pillow lavas of an old (443 Ma) ophiolite that has suffered pervasive lower greenschist facies metamorphism and deformation. X-ray mapping of initial alteration zones shows the remains of organic carbon associated with highly-concentrated Fe and S. Bioproduction of CO2 is further reflected in the low δ13C values of calcite extracted from pillow rims, compatible with microbe-induced fractionation during oxidation of organic matter. We attribute these effects to growth of sulphate-reducing bacteria at the early stage of ophiolite formation. During energy metabolism these bacteria reduce sulphate to H2S and oxidize organic matter to CO2 . Hydrogen sulphide will eventually react with iron and form pyrite, and carbon dioxide is precipitated as calcium carbonate. The results of this study may thus trigger the search for bio-signatures in glassy volcanic rocks of any age.