A quantitative and qualitative assessment using molecular markers derived from compound-specific indices for indicating groundwater impacted by petroleum spills in an oil field was recently undertaken and demonstrated serious contamination, with both high total petroleum hydrocarbons (TPH) (3.68–7.32 mg/L) and hazardous compounds in the groundwater. A petroleum source was identified, and the analysis revealed a decreasing trend of fresh petroleum input, along with groundwater advection and an increasing trend of biodegradation potential at locations farther from the source. This was confirmed via microbial analysis with both biodegrading microorganisms and diversity indices (Shannon, Simpson, and Pielou) and the principal component analysis (PCA) modeling approach, which classified the field samples into three types according to the distribution correlations between different organic compounds. Biodegradation was believed to be the dominant sink of hydrocarbons due to the increasing Pr/C17 and Ph/C18 values with seasonal changes. Raised temperatures activated the microbial degradation process; specifically, low-weight hydrocarbons degraded more rapidly than high-weight hydrocarbons, resulting in the accumulation of an unresolved complex mixture of bioproducts at locations that were farther away. Spatially, the Pr/C17 and Ph/C18 values increased from the upstream to the downstream areas, showing substantial biodegradation. The relationships between the molecular markers and chemical indices were quantified via canonical correlation analysis (CCA) to visually explain the interactive reaction processes. It was also demonstrated that the biodegradation of petroleum organics can be characterized by the consumption of dissolved oxygen and a decreasing Pr/Ph ratio, due to system reduction. These results demonstrate that compound-specific molecular markers, coupled with biochemical parameters, can effectively support a better understanding and effective fingerprinting of the fate and transport of petroleum organic contaminants, thus offering valuable technical support for a cost-effective remediation strategy.