Selenium nanoparticles (Se‐NPs) were produced adopting an environmentally benign green synthetic approach from the waste biomaterial obtained from the apple peel extract (APE). The produced NPs were characterized through UV–Visible spectroscopy (UV–Vis), atomic force microscopy (AFM), Fourier‐transform infrared spectroscopy (FT‐IR), X‐ray diffraction (XRD), field emission‐scanning electron microscopy (FE‐SEM), and transmission electron microscopy (TEM). The UV–Visible spectra exhibited the absorption peak λmax at 295 nm, confirming the formation of Se‐NPs. The AFM examination revealed the roughness and distribution of the nanoparticles, and FT‐IR ascertained the surface‐contained functional groups presence. The XRD confirmed the crystalline nature of the sample, and FE‐SEM validated the surface morphology and the size range of 52 to 84 nm. The TEM demonstrated the spherical shapes of the Se‐NPs with a face‐centered cubic crystal nature, whereas the size distribution analysis showed mean size of 70 nm. The minimum inhibitory concentration (MIC) at 500 μg mL−1 against Candida spp., namely, C. albicans, C. guilliermondii, and C. ciferrii, confirmed their anti‐fungal activity with the in control and minor statistical difference (p < 0.05) in the dissemination mean zones of inhibition for the Se‐NPs treatment against these fungi. The Se‐NPs effects on MexA gene expression demonstrated the efflux pump role in the anti‐fungal activity and gene's down regulation for the treated fungal strains. The Se‐NPs exhibited their anti‐fungal activity against the multi‐drug resistant microbes.