Every year, rivers introduce a staggering amount of hundred kilotons of plastic into the Oceans. This plastic is inhabited by microorganisms known as the plastisphere, which can be transferred between different ecosystems through the transport of microplastics. Here, we simulated the microbial colonization of polyethylene-based plastic pellets that are classically used to manufacture large-scale plastic products. The pellets were immersed for 1month in four to five sampling stations along the river-to-sea continuum of nine of the major European rivers. This study presents the first untargeted metabolomics analysis of the plastisphere, by using ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). The plastisphere metabolomes were similar in the Rhine and Rhone rivers, while being different from the Tiber and Loire rivers, which showed greater similarity to the Thames and Seine rivers. Interestingly, we found a clear distinction between plastisphere metabolomes from freshwater and marine water in most of the river-to-sea continuum, thus suggesting a complete segregation in plastisphere metabolites that is not consistent with a major transfer of microorganisms between the two contrasted ecosystems. Putative annotations of 189 discriminating metabolites suggested that lipid metabolism was significantly modulated. These results enlightened the relevance of using environmental metabolomic as complementary analysis to the current OMICs analysis.