A facile one-pot and effective green process for biogenic selenium nanoparticles (SeNPs) was obtained using the cellfree extracts of a novel yeast Magnusiomyces ingens LH-F1. The corresponding absorption peak of SeNPs was observed at ~ 560 nm by UV-vis spectrophotometer. In the present study, SeO 2 2 mM, protein 500 mg L −1 and pH 7 were preferable to the biosynthesis of SeNPs. The effects of pH, SeO 2 concentration and protein concentration on the synthesis process were different. Transmission electron microscopy image exhibited that all the SeNPs were spherical and quasi-spherical with the diameters mainly distributed in 70-90 nm (average particles size was 87.82 ± 2.71 nm). X-ray diffraction suggested that the nanoparticles were composed of standard hexagonal crystalline Se with high purity. Fourier transform infrared spectroscopy indicated that some biomolecules such as hydroxyl, carboxyl and amino groups in the yeast cell-free extracts might be involved in the formation of SeNPs. Analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that two proteins with low molecular weight approximately ~ 16 and ~ 21 kDa were detected on the surface of SeNPs and in the extracts, which could play the role of natural stabilizers and confer stability to synthesized SeNPs; whereas, unbound proteins on the SeNPs surface could act as reducing agents. Antibacterial analysis showed that the SeNPs could inhibit Arthrobacter sp. W1 (Gram positive) but not E. coli BL21 (Gram negative), which could provide reference for antimicrobial application of biogenic SeNPs.