<p>Nosocomial infections pose a major global threat to patient safety, leading to longer hospital stays, increased disability and death, higher healthcare costs, and contributing to antimicrobial resistance. These infections are often linked to inadequate disinfection, invasive medical procedures, and microbial buildup on healthcare surfaces and devices. Therefore, developing new strategies to prevent or reduce bacterial colonization and biofilm formation in healthcare settings is essential. A promising approach involves adding antibacterial metal-based nanoparticles into materials to create nanocomposites with antibacterial properties. However, their cytotoxicity to human cells remains a significant concern. Current research aims to balance antibacterial effectiveness with decreased toxicity to human cells. This study provides a comparative in vitro analysis of the cytotoxic effects of seven commonly used metal-based commercial nanoparticles: Ag, ZnO, TiO₂, CeO₂, MgO, ZrO₂, and Bi₂O₃. We evaluated their effects on bacteria related to nosocomial infections (<i>E. coli</i>,<i> S. aureus</i>,<i> P. aeruginosa</i>, and <i>S. mutans</i>) as well as relevant human eukaryotic cells (osteoblasts, fibroblasts, keratinocytes, and adipose-derived mesenchymal stem cells). Additionally, we characterized the nanoparticles’ chemical composition, size, shape, surface area, zeta-potential, hydrodynamic radius, and crystalline structure, along with the pH and conductivity of their aqueous suspensions. Our findings identify nanoparticle types and concentrations that offer optimal cytocompatibility and antibacterial activity, providing crucial guidance for developing safer and more effective antibacterial materials for targeted clinical applications.</p>