Green synthesis has recently attracted extensive attention from scientists all over the world for the production of metal nanoparticles. Selenium nanoparticles (Se NPs) have been demonstrated as a suitable supplement nutrient for the replacement of selenium ions in terms of safety and efficiency. This work presented a friendly and facile approach to synthesize the Se NPs using polyphenols content in the Cleistocalyx operculatus (CO) leaves extract. The synthesizing conditions were optimized to obtain the Se NPs with uniform distribution and shape. The prepared Se NPs were well-characterized using scanning electron microscopy, X-ray diffraction, energy diffractive spectroscopy, and Fourier-transform infrared spectroscopy. The resultant Se NPs were in spherical shape with the particle size in a range from 50–200 nm. The antimicrobial properties of Se NPs were investigated against Echerichia coli and Staphylococcus aureus, which showed reasonable activity. The acute oral toxicity of Se NPs in mice was also studied. The result indicated that Se NPs exhibited lower toxicity than that of SeO2 with the lethal concentration (50% death of mice) of 7.75 mg kg−1.