Protein corona formation on nanoparticles (NPs), affect NP physicochemical properties, cellular uptake and toxicity, and has been reported extensively. To date, studies of the occurrence and potential importance of small molecule (metabolite) coronas are limited. We sought to determine such a corona using high-sensitivity metabolomics combined with a well-established model system for freshwater ecotoxicology (Daphnia magna feeding on Chlorella vulgaris) and amino functionalised polystyrene NPs (NH2-pNPs). Initially, we optimised our method using a targeted LC-MS/MS approach for sodium dodecylsulphate (SDS) as an analogue to signalling molecules that are known to occur in our freshwater model system. Following, we performed an untargeted discovery metabolomics study – using high-sensitivity nanoelectrospray direct infusion mass spectrometry (DIMS) for the unbiased assessment of the metabolite corona of NH2-pNPs in the freshwater model system. Untargeted DIMS metabolomics reproducibly detected 100s of small molecule peaks extracted from the NH2-pNPs when exposed to conditioned media from the D. magna-C. vulgaris model system. Attempts to annotate these extracted metabolites, including through the application of van Krevelen and Kendrick Mass Defect plots, indicate a diverse range of metabolites that were not clustered into any particular class. Overall, we demonstrate the existence of an ecologically relevant metabolite corona on the surface of NPs through application of a high-sensitivity, untargeted mass spectrometry metabolomics workflow.