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Aureobasidium pullulans was grown in liquid culture media amended with selenite and tellurite and selenium (Se) and tellurium (Te) nanoparticles (NPs) were recovered after 30 d incubation. A separation method was applied to recover and characterise Se and Te NPs by asymmetric flow field flow fractionation (AF4) with online coupling to multi-angle light scattering (MALS), ultraviolet visible spectroscopy (UV-Vis), and inductively coupled plasma mass spectrometry (ICP-MS) detectors. Additional characterisation data was obtained from transm