Due to a historic focus on the underlying Cooper Basin and a growing demand for energy in Australia, a reassessment of the Eromanga Basin in central and eastern Australia is necessary, including a more detailed characterization of its Jurassic-Cretaceous petroleum system elements. The Jurassic Birkhead Formation and the Cretaceous Murta Formation are considered the most important source rocks of the Eromanga Basin. To study the petroleum generation potential of these two formations, a set of 55 rock cores from eight petroleum exploration wells was analysed. The sample set was subject to multiple-organic geochemical and petrographical analyses to evaluate organic richness, hydrocarbon potential (Rock–Eval S1 and S2), maceral composition, thermal maturity (e.g. vitrinite reflectance), and saturated and aromatic biomarker composition. The investigated fluvio-lacustrine siltstones and carbonaceous shales exhibit low to excellent total organic carbon (TOC) content, very low sulphur and low inorganic carbon content. Thermal maturity of both formations is at the onset of the oil window (immature to early mature). The Cretaceous Murta Formation shows good hydrocarbon generation potential and, in part, high production indices, while the Jurassic Birkhead Formation generally shows low kerogen to bitumen conversion and a good to very good hydrocarbon generation potential. Dispersed, rather coarse organic matter of terrigenous origin in fine-grained siliciclastic matrix with well-preserved plant remnants including “cutinite flames” is typical. Liptinite (mainly lamalginite) content is generally higher in the Murta Formation (including the occurrence of Botryococcus green algae), while vitrinite is clearly dominant in the Birkhead Formation. The Birkhead solvent extracts exhibit a more heterogeneous distribution of n-alkanes with distinct maxima in the long-chain range, whereas shorter-chain n-alkanes dominate the Murta extracts. Based on their quality and quantity of incorporated organic matter as well as thermal maturity, the petroleum generation potential of the Murta and Birkhead formations is discussed in detail.