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The Jamnagar sub-basin of Saurashtra, Gujarat is considered geologically prospective for hydrocarbons by Directorate General of Hydrocarbons (DGH), India. However the major part of the Jamnagar sub-basin is covered by Deccan Traps, hindering the exploration of Mesozoic hydrocarbon targets. In India, significant hydrocarbon finding in the stratigraphic sequence has not been established, as the major part of the Mesozoic sediments is underlying the Deccan Traps. Detection and mapping of the Mesozoic sediments below the Deccan Trap has been a long-standing complex geophysical problem facing the oil industry. The vast sheet of volcanic cover, which overlies the Mesozoic sediments acts as a geophysical shield and inhibit the effective use of conventional seismic techniques. Surface geochemical techniques based on hydrocarbon microseepage from subsurface accumulations, have potential to evaluate the prospects for petroleum exploration. In view of this, surface geochemical survey for adsorbed soil gas and microbial analysis was conducted in Jamnagar sub-basin, total 150 near surface soil samples were collected for the evaluation of hydrocarbon resource potential of the Basin. Microbial analysis revealed high bacteria counts for methane (1.32×106 cfu/gm), ethane (8.50× 105 cfu/gm), propane (6.86×105 cfu/gm) and butane oxidizing bacteria (5.70×105 cfu/gm) in soil samples of Jamnagar sub-basin. The bacterial concentration distribution maps show three distinct anomalies in the study area, indicating hydrocarbon microseepage at these places. The geochemical investigations in soil samples showed the presence of high adsorbed hydrocarbon gases concentration of methane (C1) 1 to 518 ppb and Ethane plus hydrocarbons (C2+) 1 to 977 ppb respectively. Further, carrying out integrated geo-microbial and geo-chemical studies with adsorbed soil gas showed the anomalous hydrocarbon zones nearby Khandera, Haripur, and Laloi areas which could probably aid to assess the true potential of the Basin. On the basis of microbial prospecting and adsorbed soil gas studies Jamnagar sub-basin study area appears to be a warm zone for hydrocarbon prospects and needs detailed investigation with other geo-scientific methods. Integrated geophysical studies have shown that Jamnagar sub-basin of Saurashtra has significant sediment thickness below the Deccan Traps and can be considered for future hydrocarbon exploration. The corroboration of adsorbed light gaseous hydrocarbons and hydrocarbon oxidizing bacteria suggests its efficacy as one of the potential tool in surface geochemical exploration of hydrocarbons.