Aims:  Characterization of microbial communities present in a surface petroleum seep in which hydrocarbons have been biodegraded for thousands of years in order to improve the understanding on natural petroleum biodegradation. Methods and Results:  DNA was extracted from a natural, surface petroleum seep and subjected to culture independent analysis (rRNA gene‐based denaturing gradient gel electrophoresis and phylogenetic analysis of clone libraries). Molecular analysis suggested dominance by acidophilic bacteria, especially Alphaproteobacteria (mainly bacteria related to Acidiphilium and Acidocella). Archaea were not detected, but fungi were present. pH of the samples was around 3·5. Conclusions:  Acidophilic microbial communities are associated with an acidic petroleum seep. Significance and Impact of the Study:  Microbial community structure analysis gives information on the environmental conditions under which petroleum biodegradation occurs. This knowledge could be applied to define conditions for specific cultivation or activity measurements. The activity of acidophilic micro‐organisms deserves more attention with respect to their involvement in natural petroleum degradation. This knowledge will contribute to the design of oil bioremediation strategies for polluted acidic settings.