Biosynthesis of nanoparticles included many strategies including attractive methods using a fungal medium as reducing and a capping mediator. Creation of nontoxic metal nanoparticles (NPs) pushes these materials to use a high-efficiency broad spectrum in many fields. Some problems are associated to the limited stability besides cytotoxic properties of some NPs in the biological and environmental systems; doping considered a promising solution to overcome these problems. During this investigation, green and ecofriendly synthesis approach was applied to prepare zinc oxide NP-doped selenium oxide (ZnO@SeO) NPs via mycosynthesis using Alternaria alternata. The produced ZnO@SeO NPs were characterized via physiochemical techniques including both transmission and scan electron microscope, UV-visible, X-ray diffraction, attenuated total reflectance-Fourier transform infrared, energy dispersive X-ray analyses, and dynamic light scattering. The mycosyntheis ZnO@SeO NPs were performed as a nanostructure with a size around 50 nm. Moreover, the NP formula exhibited an excellent homogeneity and stability that offered this formula to use in many applications with high performance and low aggregation ability. Great inhibitory activity of ZnO@SeO NPs was documented with inhibitory zone 28.33 mm against Staphylococcus aureus, 27.33 mm against Bacillus subtilis, 27.67 mm against Bacillus cereus, 27.17 mm against Pseudomonas aeruginosa, 27.5 mm against Escherichia coli, 24.33 mm against Salmonella typhi, 23.33 mm against Candida albicans, and 18.17 mm against Aspergillus niger compared to undoped ZnONPs or antibiotic/antifungal. Less effectiveness of ZnO@SeO NPs was observed on normal cells (Vero cell line CCL-81) compared with ZnONPs, while more effectiveness was observed on cancerous cell lines MCF7. ZnO@SeO NPs were more effective for methylene blue photodegradation than undoped ZnONPs with 83.1% and 35.1%, respectively at 300 min.