In the light of the fast growing several applications of selenium nanoparticles (SeNPs) in different industrial and agricultural sectors, this paper was conducted to explore the suitability of endophytic fungi as nano-factories for SeNPs. Thus, 75 fungal isolates were recovered from plant tissues and tested for their efficacy to biosynthesize SeNPs. Four promising strains were found able to synthesis SeNPs with different characteristics and identified. These strains were Aspergillus quadrilineatus isolated from the twigs of Ricinus communis, Aspergillus ochraceus isolated from the leaves of Ricinus communis, Aspergillus terreus isolated from the twigs of Azadirachta indica, and Fusarium equiseti isolated from the twigs of Hibiscus rose-sinensis. The synthesized SeNPs were characterized by several techniques viz., UV–Vis, X-ray diffraction, Dynamic light scattering analyses, High resolution transmission electron microscopy, and Fourier transform infrared spectroscopy, to study their crystalline structure, particle sized distribution, and morphology. Furthermore, the in vitro antimicrobial and antioxidant activities were evaluated. SeNPs synthesized by the four strains showed potent antifungal and antibacterial potentials against different human and phyto- pathogens. Moreover, SeNPs synthesized by the respective strains showed promising antioxidant power with IC50 values of 198.32, 151.23, 100.31, and 91.52 µg mL− 1. To the best of our knowledge, this is the first study on the use of endophytic fungi for SeNPs’ biosynthesis. The presented research recommends the use of endophytic fungi as facile one-pot production bio-factories of SeNPs with promising characteristics.