This work reports a simple and green approach for synthesizing selenium nanoparticles (SeNPs) dispersed in glycerol, using ascorbic acid as a reducing agent. The formation of SeNPs in glycerol was monitored by Ultraviolet visible spectroscopy (UV-vis), while the characteristics of SeNPs were provided by X-ray diffraction (XRD), Fourier transmission infrared spectroscopy (FT-IR), energy dispersive X-ray (EDX) mapping analysis, and high-resolution transmission electron microscopy (HR-TEM). The zero-valent SeNPs were spherical and remarkably small, averaging 3.9 nm in diameter. Significantly, the colloidal SeNPs solution in glycerol exhibited stability for at least two months without any aggregation signs, a stark contrast to selenium precipitated in water. A comparative study with other polyols (ethylene glycol and propylene glycol) identified glycerol as uniquely effective, likely because its supramolecular structure promotes superior SeNPs dispersion. The glycerol-dispersed SeNPs demonstrated significant inhibitory efficacy against the growth of both S. aureus and E. coli bacteria. This finding suggests their substantial potential for diverse applications in antibacterial and biomedical domains.