Tainá Pereira da Silva Oliveira, Alan Kelbis Oliveira Lima, Talita Pereira Gonçalves, Isadora Florêncio de Souza, Sônia Nair Báo, Namuhell Oliveira da Silva, Patrícia Albuquerque, Ildinete Silva-Pereira, Luís Alexandre Muehlmann
Background/Objectives: Optimizing synthesis parameters is essential to ensure the quality and stability of nanostructures. This study aimed to optimize the synthesis of selenium nanoparticles (SeNPs) by chemical reduction, using sodium selenite (Na2SeO3), ascorbic acid (AA), and polyvinyl alcohol (PVA) at different concentrations, volumes, and molar ratios. The effects of reduction time, purification steps, and variations in the concentration of the precursor and reducing agent, as well as in the volume of the stabilizer, on the characteristics of SeNPs were investigated to ensure their long-term stability, maintenance of their properties, and biological applicability. Methods: The SeNPs were analyzed by UV/Vis absorption spectroscopy, Dynamic Light Scattering (DLS), and Transmission Electron Microscopy (TEM), and were also evaluated for antifungal activity against the SC5314 strain of Candida albicans. Results/Conclusions: Monodisperse SeNPs were obtained under high concentrations of Na2SeO3 and AA, short reduction time, higher volumes of PVA (2–4 mL), and purification at 24.300× g, presenting a spherical morphology, hydrodynamic diameter of 137.0–171.7 nm, dry diameter of 20–120 nm, polydispersity index of 0.049–0.306, Zeta potential of −7.79 to −19.6 mV, and stability for up to 180 days. In the absence or presence of 1 mL of PVA, the SeNPs were predominantly amorphous. Regarding biological activity, the SeNPs did not exhibit antifungal activity under the experimental conditions in the tested strain. Together, this study provides a comprehensive update on the synthesis of SeNPs under different conditions and their stability over time, contributing to the consolidation of knowledge in the field.