Biofilms occurring in seepage groundwater contaminated with petroleum in an urban subway drainage system were characterized. The development of biofilms was observed only in the sites where petroleum-contaminated groundwater had seeped or was seeping. Moreover, the conditions of the biofilms such as color and development extent were influenced by the amount of spilled petroleum: By increasing the amount of spilled petroleum, the amount of biofilms increased and its color whitened. It deteriorated and became dark-brown if the contaminated groundwater did not seep any more. These facts indicate that the biofilms can be used as a preliminary indicator to identify the locations of fuel contaminated sumps and seeps without a more detailed assessment such as instrumental analysis. The biofilms were capable of degrading petroleum at 15°C, which is similar to the average temperature of the seepage groundwater. Filamentous bacteria, Sphaerotilus spp., were isolated from the biofilms. It is considered that these bacteria are responsible for the development of biofilms in the seepage groundwater contaminated with petroleum because they can secrete extracellular polymeric substances.