The use of nanotechnologies in the applied biomedical sciences can offer a new way to treat infections and disinfect surfaces, materials, and products contaminated with various types of viruses, bacteria, and fungi. The Cu-Au nanoparticles (NPs) were obtained by an eco-friendly method that allowed the obtaining in a one-step process of size controlled, well dispersed, fully reduced, highly stable NPs at very mild conditions, using high energy ionizing radiations. The gamma irradiation was performed in an aqueous system of Cu2+/Au3+/Sodium Dodecyl Sulfate (SDS)/Ethylene Glycol. After irradiation, the change of color to ruby-red was the first indicator for the formation of NPs. Moreover, the UV-Vis spectra showed a maximum absorption peak between 524 and 540 nm, depending on the copper amount. The Cu-Au NPs presented nearly spherical shapes, sizes between 20 and 90 nm, and a zeta potential of about −44 mV indicating a good electrostatic stability. The biocidal properties performed according to various standards applied in the medical area, in dirty conditions, showed a 5 lg reduction for Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus hirae, a 5 lg reduction for both enveloped and non-enveloped viruses such as Adenovirus type 5, Murine Norovirus, and human Coronavirus 229E, and a 4 lg reduction for Candida albicans, respectively. Thus, the radiochemically synthesized Cu-Au alloy NPs proved to have high biocide efficiency against the tested bacteria, fungi, and viruses (both encapsulated and non-encapsulated). Therefore, these nanoparticle solutions are suitable to be used as disinfectants in the decontamination of hospital surfaces or public areas characterized by high levels of microbiological contamination.