Hydrocarbon exploration has long been based on such costly and time-intensive methods as geophysical surveys, geological studies, and drilling. In recent years, however, researchers have started to consider such inexpensive alternatives as surface geochemistry for hydrocarbon exploration. Some 100 years ago, the leakage of hydrocarbons onto the surface in the form of micro- and macro-seepages motivated researchers toward drilling a well in the Khourian Desert in the south of Semnan Province, Iran. Upon drilling the well, researchers found evidences of non-released (free) hydrocarbons. These findings drove further study of the area using surface geochemistry while considering the nearby hydrocarbon accumulation in Qom Formation. Conventional and indirect surface geochemical methods provide an insight into the relationship between surface and subsurface hydrocarbons. In the present work, the results of the Rock–Eval pyrolysis showed total organic carbon (TOC) values in the range of 0.31–4.13 wt.% and S1 peaks between 0.07 and 27.35. Sulfur isotope analysis showed a sulfur isotopic value of −0.4. The study of hydrocarbon-oxidizing bacteria showed the presence of bacterial colonies in MSM at 1.22 × 106 cfu/g of soil sample. We further investigated surface changes due to the presence of free hydrocarbons and pH variations (4.9–8) resulted from the changes in the concentrations of calcium carbonate and iron. According to the results and given the presence of organic sulfur in the samples, the occurrence of Gach-i-turush and alike phenomena was proposed in this area. The results of geo-microbial prospecting method, surface secondary changes, and sulfur isotope studies were well in agreement with the characteristics of the existing hydrocarbon reserves in this area. Surface geochemical surveys can precede other geochemical and geophysical surveys to identify surface anomalies and hence focus on more probable locales of hydrocarbon accumulation in the Khourian Desert, central Iran.