Selenium nanoparticles (Se-NPs) has recently received great attention over owing to their superior optical properties and wide biological and biomedical applications. Herein, crystallographic and dispersed spherical Se-NPs were green synthesized using endophytic fungal strain, Penicillium crustosum EP-1. The antimicrobial, anticancer, and catalytic activities of biosynthesized Se-NPs were investigated under dark and light (using Halogen tungsten lamp, 100 Watt, λ > 420 nm, and light intensity of 2.87 W m−2) conditions. The effect of Se-NPs was dose dependent and higher activities against Gram-positive and Gram-negative bacteria as well different Candida spp. were attained in the presence of light than obtained under dark conditions. Moreover, the viabilities of two cancer cells (T47D and HepG2) were highly decreased from 95.8 ± 2.9% and 93.4 ± 3.2% in dark than those of 84.8 ± 2.9% and 46.4 ± 3.3% under light-irradiation conditions, respectively. Significant decreases in IC50 values of Se-NPs against T47D and HepG2 were obtained at 109.1 ± 3.8 and 70.4 ± 2.5 µg mL−1, respectively in dark conditions than 19.7 ± 7.2 and 4.8 ± 4.2 µg mL−1, respectively after exposure to light-irradiation. The photoluminescence activity of Se-NPs revealed methylene blue degradation efficiency of 89.1 ± 2.1% after 210 min under UV-irradiation compared to 59.7 ± 0.2% and 68.1 ± 1.03% in dark and light conditions, respectively. Moreover, superior stability and efficient MB degradation efficiency were successfully achieved for at least five cycles.