A natural attenuation experiment was carried out using a lysimeter for 308 days after contaminating the subsoil with hydrocarbons (HCs) and the changes in the structures of microbial community in the hydrocarbon (HC) contaminated subsoil were monitored by quinone profile analysis. The residues of HCs remained for 217 days in the subsoil after the contamination. The amount of total quinones (TQ), an indicator of microbial biomass, significantly increased in the HC contaminated subsoil for 217 days, comparing with that of the background subsoil or the subsoil before the addition of HCs. The major quinone species and the quinone composition, indicators of community structure, were significantly different between the HC contaminated soil and the background soil for 217 days. The major increased quinine species in the HC contaminated soil were menaquinone-8(H4), menaquinone-9(H2) and ubiquinone-9, indicating the propagation of Gram-positive bacteria with high guanine and cytosine content and gamma-subclass of Proteobacteria and fungi. There was no significant difference in the diversity of the quinone species (DQ), an indicator of taxonomic diversity of microbial community, except for the decrease in DQ in the shallow subsoil after 35 days when a high concentration of HCs was detected. After 308 days when the HCs in the subsoil disappeared, TQ returned to the level of the background soil, and no significant difference in quinone composition were observed between the HC contaminated soil and the background soil. The results suggested that respiratory quinones are effective biomarkers for characterizing the temporal changes of microbial community in the HC contaminated subsoil.