This present study reports a one-pot environmentally friendly synthesis and characterizations of selenium nanoparticles (SeNPs) fromLysinibacillussp. NOSK and their applications. SeNPs were subjected to microscopic and spectrophotometric analysis by uses UV-visible (UV-Vis) spectrophotometer, scanning electron microscopy (SEM), light microscope, dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA). The SEM analysis has revealed the spherical shape of SeNPs with a size approximately of 130 nm with a good polydispersity index. Moreover, SeNPs have been evaluated for their effect on the growth of pathogen bacteria. Results revealed that SeNPs significantly have an inhibitory effect on the tested bacteria. In addition to antimicrobial activity, SeNPs anti-biofilm activity was also investigated. Results from anti-biofilm analysis showed that SeNPs inhibited the biofilm ofPseudomonas aeruginosacompared to non-treated samples. Besides, cytotoxicity analysis showed that the synthesized SeNPs had no severe side effects on healthy cells even if the concentration was high. Finally, cell attachment studies which was used fabricated SeNPs doped cellulose acetate electrospun nanofibers and cytocompatibility assay demonstrated that SeNPs contributed cell growth and adhesion that is SeNPs provided a favorable environment to attach and proliferate and was bioactive. Considering all the outcomes of the studies together, our findings introduces the possibility of using SeNPs for biomedical applications.