Today, one of the biggest challenges is infections in the painted walls of hospitals. Acrylic-based paints are a target of antibiotic-resistant microorganisms since they contain cellulosic compounds as thickeners. The aim of this study was to synthesize and investigate the biocidal activity and toxicity of heterophase ZnO-Ag nanoparticles fixed in water-based acrylic paint layers in reference to a nontreated water-based paint. The ZnO-Ag nanoparticles with average particle sizes of about 80 nm were simply obtained by electrical explosion of two twisted wires in an oxygen-containing atmosphere. The nanoparticles and modified paint were characterized using SEM, TEM, XPS, and XRD techniques. The antimicrobial activity of the nanoparticles and modified paint layers was tested against P. aeruginosa, S. aureus, MRSA, E. coli bacteria, and C. albicans using ISO 22196. The antiviral activity against smallpox virus was tested according to ISO 21702. Flow cytometry tests were used to investigate the toxicity of the modified paint coating. As-synthesized nanoparticles had “Janus-like” morphology, with a clear interface inside the nanoparticle. Nanoparticles had enhanced antibacterial activity, which is based on the nanoparticle photocatalytic activity in water decomposition and reactive oxygen species generation. The paint coating with a ZnO-Ag nanoparticle mass ratio of 1.0 wt.% displayed significant antibacterial activity (more than a 99% reduction) and 100% antifungal activity. In addition, this coating inactivates >99% of the virus after 2 h of contact relative to a nontreated control paint. The paint coating showed low toxicity against the sensitive 3T3 fibroblast cell line. More than 90% cell viability was observed after 24 h of incubation with the sample extract. Therefore, heterophase ZnO-Ag nanoparticles have high biocidal activity and low toxicity use and can be applied to other commercial water-based paints to improve their performance against pathogens.