More and more studies are appearing devoted to the possibility of using nanotechnology in the prevention and treatment of neurodegenerative processes. This study aimed to develop a technology for obtaining small and large selenium nanoparticles (SeNPs) by laser ablation of a solid target. And also to study the effects of these different-sized nanoparticles on the physiology (calcium signalling, redox status, regulation of cell death) of cortical cells in culture in toxic models of ischemia-like conditions and glutamate excitotoxicity, primarily reflecting pathological processes in the brain during injuries, strokes, and neurodegenerative diseases. In the present study, a technology for obtaining SeNPs of different sizes is presented. Using molecular biology methods, fluorescence microscopy, viability assays, and measurement of ROS production in cortical cells, differences in the effects of SeNPs were revealed in acute and chronic experiments, in models of glutamate excitotoxicity and ischemia-like conditions. SeNPs with a size of 100 nm were most effective in suppressing cell death, modulating the cellular redox status, and Ca signalling system. 10 nm-sized SeNPs enhanced cell death in toxic models by upregulating genes encoding pro-inflammatory, pro-apoptotic, and pro-oxidative proteins. Thus, when creating nanoparticles to protect brain cells, the influence of the nanoparticle diameter should be taken into account, as small nanoparticles are capable of exerting a cytotoxic effect on brain cells, and such particles are more appropriate in anticancer therapy.