Specific geochemical analyses included API gravity, sulfur content, gas chromatography, and the advanced gas chromatography-mass spectrometry for biomarker distributions, in conjunction with stable carbon isotope geochemistry, to characterize the depositional environmental conditions and maturation assessment of the studied crude oils. For this purpose ten crude oils were collected from Miocene, Upper Cretaceous, and Nubia Sandstone representing the central-southern Gulf of Suez. The results showed that two different oil types identified as Miocene oils are characterized by a dominant oleanane and low gammacerane biomarkers, suggesting an overall angiosperm land plants input of terrigenous organofacies source rock. Pre-Miocene oils are distinguished by a dominant gammacerane and low oleanane biomarker distributions, which may be generated from fully mature marine carbonate source rocks. The higher sterane isomerization parameters, C29 ss ss/(ss ss + ) and C29 20S/(S+R), further support the higher thermal maturation level for the Pre-Miocene oils rather than the Miocene type.