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Metal nanoparticles are assumed to be a new generation of biologically active materials. The integrations between more than one metal are synergetic multifunctional features. In the current study, trimetallic copper-selenium-zinc oxide nanoparticles (Tri-CSZ NPs) were successfully mycosynthesized using Aspergillus niger through an ecofriendly method for the first time. The biosynthesis of the particles was characterized using physiochemical and topographical analysis. The physiochemical analysis included Fourier transform infrared spectroscopy (FTIR), which affirmed that the biosynthesis of Tri-CSZ NPs relies on the functional groups of fungal filtrates. Additionally, the UV-visible and X-ray diffraction patterns were proposed for the formation of Tri-CSZ NPs; moreover, topography analysis confirmed that the micromorphology of the nanoparticles were similar to a stick, with ends having a tetragonal pyramid shape, and with an average nanosize of about 26.3 ± 5.4 nm. Cytotoxicity results reveled that the Tri-CSZ NPs have no cytotoxicity on the human normal cell line Wi 38 at low concentrations, where the IC50 was 521 µg/mL. Furthermore, the antifungal activity of the Tri-CSZ NPs was evaluated. The antifungal results revealed that the Tri-CSZ NPs have promising antifungal activity against Mucor racemosus , Rhizopus microsporus , Lichtheimia corymbifera , and Syncephalastrum racemosum , where the minimum inhibitory concentrations (MICs) were 1.95, 7.81, 62.5, and 3.9 µg/mL, and the minimum fungicidal concentrations (MFCs) were 250, 62.5, 125, and 1000 µg/mL, respectively. In conclusion, Tri-CSZ NPs were successfully mycosynthesized using A. niger , which have a promising antifungal activity against fungi causing mucormycosis.