Microbial communities inhabiting coastal marine sediments at intertidal zones are subjected to fluctuating environmental conditions according to tide level and reworking activities of macroorganisms. Such fluctuations are important drivers for the microbial communities’ assemblages and organization that in turn determine the microbial hydrocarbon-degradation processes. Notwithstanding the difficulties to access the entire ecosystem complexity, experimental ecology studies, involving more or less sophisticated experimental systems, have demonstrated to be suitable approaches. Several systems have been developed to tackle the role of microorganisms in hydrocarbon degradation in marine sediments in order to decipher the role of microbial communities in hydrocarbon degradation and understand the microbial behavior in response to an oil spill. We describe here a mesocosm system that maintains sediments under conditions as close as possible to those prevailing in the environment. We explain how to collect mudflat sediments conserving their structure and how to apply crude oil in order to simulate an oil spill. Subsampling procedures are also provided for following the fate of crude oil hydrocarbons, the macrofauna burrowing activity, as well as the behavior and dynamic of microbial communities.