Abstract : Faecal contaminations from human and animal wastes not only contribute to the degradation of aquatic systems but also affect water quality posing threat to human health from exposure to pathogens. To reduce these risks and improve water quality, it is necessary to develop of tools capable of identifying faecal contamination sources. This issue is exacerbated in Europe by the hardening of guidelines for bathing waters and shellfish waters and the obligation to identify and prioritize sources of contamination. The aim of this thesis is to validate the use of steroids as a tool for tracing the origin of faecal contamination in surface waters. The method is based on principal component analysis of six fecal stanols. The scientific approach was conducted in three steps, from molecular scale to the watershed scale. The specificity of steroids and the homogeneity of sources were studied on bovine and porcine manures and on influent, effluent and sludge of waste water treatment plant. Then the conservation and the persistence of the steroid fingerprint were evaluated during the transport of raw samples to the environment receiving by experiments under controlled conditions. Finally, the method developed has been validated at the scale of two watersheds impacted by recurrent multi-sources contaminations. All the results obtained in this work lead to validate the use of steroids as an operational tool to trace faecal sources contamination.