Natural hydrocarbon seepage has helped petroleum exploration as a direct indicator of gas and/or oil subsurface accumulations. Surface macro-seeps are generally an indication of a fault in an active Petroleum Seepage System belonging to a Total Petroleum System. This paper describes a case study in which multiple analytical techniques were used to identify and characterize trace petroleum-related hydrocarbons and other volatile organic compounds in groundwater samples collected from Sousse aquifer (Central Tunisia). The analytical techniques used for analyses of water samples included gas chromatography-mass spectrometry (GCMS), capillary GC with flame-ionization detection, Compound Specific Isotope Analysis, Rock Eval Pyrolysis. The objective of the study was to confirm the presence of gasoline and other petroleum products or other volatile organic pollutants in those samples in order to assess the respective implication of each of the potentially responsible parties to the contamination of the aquifer. In addition, the degree of contamination at different depths in the aquifer was also of interest. The oil and gas seeps have been investigated using biomarker and stable carbon isotope analyses to perform oil-oil and oil-source rock correlations. The seepage gases are characterized by high CH4 content, very low δ13CCH4 values (-71,9 ‰) and high C1/C1–5 ratios (0.95–1.0), light deuterium–hydrogen isotope ratios (- 198 ‰) and light δ13CC2 and δ13CCO2 values (-23,8‰ and-23,8‰ respectively) indicating a thermogenic origin with the contribution of the biogenic gas. An organic geochemistry study was carried out on the more ten oil seep samples. This study includes light hydrocarbon and biomarkers analyses (hopanes, steranes, n-alkanes, acyclic isoprenoids, and aromatic steroids) using GC and GC-MS. The studied samples show at least two distinct families, suggesting two different types of crude oil origins: the first oil seeps appears to be highly mature, showing evidence of chemical and/or biological degradation and was derived from a clay-rich source rock deposited in suboxic conditions. It has been sourced mainly by the lower Fahdene (Albian) source rocks. The second oil seeps was derived from a carbonate-rich source rock deposited in anoxic conditions, well correlated with the Bahloul (Cenomanian-Turonian) source rock., {"references":["Link, W.K., 1952. Significance of oil and gas seeps in world oil\nexploration. American Association of Petroleum Geologists Bulletin 36,\n1505–1540.","Jones, V.T., Drozd, R.J., 1983. Predictions of oil or gas potential by\nnear-surface geochemistry. American Association of Petroleum\nGeologists Bulletin 67, 932–952.","Abrams, M.A., 2005. Significance of hydrocarbon seepage relative to\npetroleum generation and entrapment. Marine and Petroleum Geology\n22, 457–477.","Etiope, G., Feyzullayev, A., Baciu, C.L., 2009a. Terrestrial methane\nseeps and mud volcanoes:a global perspective of gas origin. Marine and\nPetroleum Geology 26, 333–344.","Sami Khomsi, MouradBédir, Mohamed Soussi, Mohamed Ghazi Ben\nJemia, Kmar Ben Ismail-Lattrache., 2006. Mise en évidence en\nsubsurface d'événements compressifs en Tunisie orientale (Sahel) :\ngénéralité de la phase atlasique en Afrique du Nord. C. R. Geoscience\n338 (2006) 41–49.","Layeb M. (1991):Etude géologique, géochimique et minéralogique\nrégionale des faciès riches en matière organique de la Formation Bahloul\nd'âge Cénomano-Turonien dans le domaine de la Tunisie Centrale.\nThèse de Doctorat de spécialité, Faculté des Sciences de Tunis, 209p.","Saidi M. and Inoubli H. (2001): Geochemistry and organic petrography\nof proven Tunisian source rocks (poster). 20th International Meeting of\nOrganic geochemistry (Nancy 2001).","Saidi M. (2003): Tunisian source Rocks and oils\nGeochemicalcharacteristics and Oil-Source rock correlation. Rapport\ninterne ETAP.","Saidi M. (1993): Etude géologique et géochimique des roches mères\nalbo-vraconiennes dans le domaine de la Tunisie Centro-Septentrionale.\nThèse de Doctorat de sépcialité, Université de Tunis II, 135p.\n[10] A. Belhaj Mohamed, M. Saidi and B. Ibrahim., 2014. Migration\nPathway Evaluation through Analysis of Hydrocarbon Seeps in the\nPelagian basin, Sahel of Tunisia. 76th EAGE Conference and Exhibition\n2014\n[11] Tissot, B.P., Welte, D.H., 1984. Petroleum Formation and Occurrence,\nsecond ed. Springer-Verlag, Berlin, 235 pp.\n[12] Schoell, M., 1980. The hydrogen and carbon isotopic composition of\nmethane from natural gases of various origins. Geochimica et\nCosmochimica Acta 44, 649–662.\n[13] Schoell, M., 1983. Genetic characterization of natural gases. American\nAssociation of Petroleum Geologists Bulletin 67, 2225–2238.\n[14] Whiticar, M.J., Faber, E., Schoell, M., 1986. Biogenic methane\nformation in marine and fresh water environments: CO2-reduction vs.\nacetate fermentation–Isotope evidence. Geochimica et Cosmochimica\nActa 50, 693–709.\n[15] Thompson, K. F. M., 1983, Classification and thermal history of\npetroleum based on light hydrocarbons: Geochimica et Cosmochimica\nActa, v. 47, p. 303–316, doi:10.1016/0016-7037(83)90143-6."]}