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 transmission electron microscopy (TEM), and dynamic light scattering (DLS). Solutions of 0.2% Novachem surfactant and 10 mM phosphate buffer were compared as mobile phases to investigate optimal AF4 separation and particle recovery using Se-NP as a model sample. 88% recovery was reported for 0.2% Novachem solution, compared with 50% recovery for phosphate buffer. Different crossflow (Cflow) rates were compared to further investigate optimum separation, with recoveries of 88% and 30% for Se-NPs, and 90% and 29% for Te-NPs for 3.5 mL min-1 and 2.5 mL min-1 respectively. Zeta-potential (ZP) data suggested higher stability for NP elution in Novachem solution, with increased stability attributed to minimised NP-membrane interaction due to PEGylation. Detection with MALS showed monodisperse Se-NPs (45-90 nm) and polydisperse Te-NPs (5-65 nm).Single particle ICP-MS showed mean particle diameters of 49.7 ± 2.7 nm, and 135 ± 4.3 nm, and limit of size detection (LOSD) of 20 nm and 45 nm for Se-NPs and Te-NPs respectively. TEM images of Se-NPs and Te-NPs displayed a spherical morphology, with the Te-NPs showing a clustered arrangement, which suggested electrostatic attraction amongst neighbouring particles. Particle hydrodynamic diameters (dH) measured with dynamic light scattering (DLS) further suggested monodisperse Se-NPs and polydisperse Te-NPs distributions, showing good agreement with AF4-MALS for Se-NPs, but suggests that the Rg obtained from AF4-MALS for Te-NP was unreliable. The results demonstrate a complementary application of asymmetric flow field-flow fractionation (AF4), ICP-MS, light scattering, UV-Vis detection, and microscopic techniques to characterise biogenic Se and Te NPs.