Herein, four endophytic fungal strains living in healthy roots of garlic were used to produce selenium nanoparticles (Se-NPs) via green synthesis. Penicillium verhagenii was found to be the most efficient Se-NPs producer with a ruby red color that showed maximum surface plasmon resonance at 270 nm. The as-formed Se-NPs were crystalline, spherical, and well-arranged without aggregation, and ranged from 25 to 75 nm in size with a zeta potential value of -32 mV, indicating high stability. Concentration-dependent biomedical activities of the P. verhagenii-based Se-NPs were observed, including promising antimicrobial activity against different pathogens (Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Candida albicans, C. glabrata, C. tropicalis, and C. parapsilosis) with minimum inhibitory concentration (MIC) of 12.5-100 µg mL. The biosynthesized Se-NPs showed high antioxidant activity with DPPH-scavenging percentages of 86.8 ± 0.6% at a concentration of 1000 µg mL and decreased to 19.3 ± 4.5% at 1.95 µg mL. Interestingly, the Se-NPs also showed anticancer activity against PC3 and MCF7 cell lines with IC of 225.7 ± 3.6 and 283.8 ± 7.5 µg mL, respectively while it is remaining biocompatible with normal WI38 and Vero cell lines. Additionally, the green synthesized Se-NPs were effective against instar larvae of a medical insect, Aedes albopictus with maximum mortality of 85.1 ± 3.1, 67.2 ± 1.2, 62.10 ± 1.4, and 51.0 ± 1.0% at a concentration of 50 µg mL for I, II, III, and IV-instar larva, respectively. These data highlight the efficacy of endophytic fungal strains for cost-effective and eco-friendly Se-NPs synthesis with different applications.