The Late Oligocene Mequinenza formation (SE Ebro Basin, NE Spain) records the development of extensive, shallow, low-gradient lacustrine zones, which evolved under a subtropical, warm climatic regime, affected by humidity–aridity cycles. Carbonate-dominated inner lacustrine sequences, which include thin coal seams, are laterally related to muddy carbonate and siliciclastic marginal lacustrine assemblages where evaporites occur. Water solute concentration of the lacustrine zones changed during the alternative high- and low-stand episodes, with lacustrine waters ranging mainly from fresh to oligosaline. Nonsulphurised (NSC) and sulphurised (OSC) organic compounds occur in the inner lacustrine facies and record significant contributions from aquatic and terrestrial higher plants, bacteria (cyanobacteria, photosynthetic anaerobic bacteria) and algae. These biomarkers show that the lacustrine system was eutrophic and the water column was poorly oxygenated and sometimes stratified, as evidenced by occasional finely laminated facies and gammacerane. Organic matter S/C ratio (lower in carbonate than in coal and coal-related facies) suggests significant variations in the lacustrine autotrophic production and/or the allochthonous organic matter contribution. Higher plant biomarkers were recorded mainly as normal, nonsulphurised lipids. By contrast, bacterial and algal lipids (hopanoids, steroids and isoprenoids), under intense sulphate reduction conditions, incorporated sulphur in their hydrocarbon skeleton and gave rise to OSC with distributions of thienyl and thiolanylhopanoids, epithiosteranes and polyprenoids. Linear thiols are new biomarkers, which probably were generated by the early incorporation of sulphur into funtionalised precursors (e.g., alkanols). Intense methanogenesis was enhanced in some lacustrine zones owing to high organic matter input and, as a consequence, biomarkers of methanotrophic and methylotrophic bacteria, related to methane emission, are also found.