Microbial enhanced oil recovery (MEOR) represents one of the most significant technologies in the tertiary stage of oil recovery in the present era. This technology, which is cost-effective and environmentally benign, is regarded as one of the most promising technologies for oil recovery. The objective of this study is to elucidate the impact of functional microorganisms on crude oil during the implementation of this technology. Firstly, the crude oil components from an oilfield in China were analyzed by liquid–solid adsorption chromatography, simulated distillation and gas chromatography. Subsequently, gas chromatography-mass spectrometry was employed to detect the crude oil components before and after microbial action to ascertain the utilization efficiency to different components and carbon numbers of crude oil. Finally, the intermediate metabolites of the microbial degradation of n-eicosane were analyzed to reveal the metabolic pathway of long-chain alkane. This study offers technical and theoretical support for researching the metabolic mechanism of functional microorganisms in oil reservoirs, which is of great significance for enhanced microbial oil recovery efficiency and remediation of oil pollution.