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Mercury, Hg, is one of the most harmful elements present in the Earth, and has both natural and anthropological sources. Moreover, Hg undergoes many different transformation pathways during its biogeochemical, or industrial, cycles which in general involve redox reactions, both abiotic and biotic, and phase changes. Despite the toxicity of this pollutant, there is still a lack in the knowledge about its biogeochemistry in the ecosystem and, therefore, it is of utmost relevance to develop new scientific approaches to deepen its transformation mechanisms and to identify the contamination sources. In this context, the determination of mercury stable isotopes ratios seems to be an extremely interesting and challenging application to verify the “provenance” of the element, i.e. to identify whether it is of natural or anthropogenic source. This approach can be useful in case of polluted areas where many are the contamination sources in order to plan an environmental requalification, as in the present case study represented by the National Interest Site of the lagoon of Marano-Grado (Trieste, Italy), which is the main object of the present investigation.