Bitumens extracted from 2.7 to 2.5 billion-year-old (Ga) shales of the Fortescue and Hamersley Groups in the Pilbara Craton, Western Australia, contain traces of molecular fossils. Based on a combination of molecular characteristics typical of many Precambrian bitumens, their consistently and unusually high thermal maturities, and their widespread distribution throughout the Hamersley Basin, the bitumens can be characterized as 'probably of Archean age'. Accepting this interpretation, the biomarkers open a new window on Archean biodiversity. The presence of hopanes in the Archean rocks confirms the antiquity of the domain Bacteria, and high relative concentrations of 2α-methylhopanes indicate that cyanobacteria were important primary producers. Oxygenic photosynthesis therefore evolved > 2.7 Ga ago, and well before independent evidence suggests significant levels of oxygen accumulated in the atmosphere. Moreover, the abundance of cyanobacterial biomarkers in shales interbedded with oxide-facies banded iron formations (BIF) indicates that although some Archean BIF might have been formed by abiotic photochemical processes or anoxygenic phototrophic bacteria, those in the Hamersley Group formed as a direct consequence of biological oxygen production. Biomarkers of the 3β-methylhopane series suggest that microaerophilic heterotrophic bacteria, probably methanotrophs or methylotrophs, were active in late Archean environments. The presence of steranes in a wide range of structures with relative abundances like those from late Paleoproterozoic to Phanerozoic sediments is convincing evidence for the existence of eukaryotes in the late Archean, 900 Ma before visible fossil evidence indicates that the lineage arose. Sterol biosynthesis in extant eukaryotes requires molecular oxygen. The presence of steranes together with biomarkers of oxygenic photosynthetic cyanobacteria suggests that the concentration of dissolved oxygen in some regions of the upper water column was equivalent to at least ∼1% of the present atmospheric level (PAL) and may have been sufficient to support aerobic respiration.