The Vienna Basin, a major petroleum province in Central Europe, hosts hydrocarbons in stacked carbonate and siliciclastic reservoirs. The study of 84 oil and 51 gas samples in the Austrian sector yields new insights into the petroleum system. The Upper Jurassic Mikulov and Falkenstein formations are the only significant source rocks, which generated oil between 0.7 and 1.0%Rr, and gas between 1.1 and 1.6%Rr. Microbial gas prevails in the southern part of the basin. Biodegradation affects oil down to a depth of 2000 m. Miocene reservoirs in the hanging wall of major faults and oils in transgressive sands are more prone to biodegradation than footwall flysch reservoirs and oils in turbiditic sands in structural-stratigraphic traps. Anaerobic biodegradation results in the formation of isotopically heavy CO2 and isotopically light (secondary) microbial methane. Hydrocarbons in deep carbonate reservoirs are affected by thermochemical sulphate reduction (TSR). While TSR-affected gas is rich in H2S and CO2, TSR-affected oil is characterized by increased dibenzothiophene/phenantrene (DBT/Ph) ratios. In clastic reservoirs, H2S is removed by pyrite precipitation, whereas DBT/Ph ratios remain high. Hence, high DBT/Ph ratios may be used as proxy for TSR. Stable sulphur isotopes signatures confirm Upper Triassic anhydrites as the main sulphur source for H2S.