The present study focuses on the biogenic synthesis of selenium nanoparticles (Se NPs) using Pelargonium zonale leaf extract under microwave irradiation. Response surface methodology was used to evaluate the effects of the synthesis parameters, namely amounts of the leaf extract (0.5–2.5 ml) and amounts of the 10 mm sodium selenite solutions (15–65 ml), at constant microwave heating (4 min), on the concentration and particle size of the fabricated Se NPs, optimize the synthesis conditions and verify the generated models and the procedures. The obtained results indicated that Se NPs with preferable attributes of mean particle size (50 nm), zeta potential (−24.6 mV), absorbance [34.6% absorbance units (a.u.)] and broad absorption peak (319 nm) were formed at the optimum synthesis conditions including amounts of 1.48 ml and 15 ml Pelargonium leaf extract and sodium selenite solution, respectively. The antibacterial activities of the synthesized Se NPs against Escherichia coli and Staphylococcus aureus indicated that the created NPs had higher antibacterial activities toward the Gram-positive bacteria. Furthermore, the synthesized Se NPs indicated higher antifungal activities against Colletotrichum coccodes and Penicillium digitatum.